Circulating CD4+ CD25+ regulatory T cells correlate with chronic hepatitis B infection

Abstract

Circulating CD4+ CD25+ regulatory T cells (Tregs) have been demonstrated to maintain immunotolerance and suppress the antigen-specific or antigen-non-specific T-cell responses, but their role in chronic hepatitis B (CHB) infection in humans has not been well characterized. In this study, we analysed the frequency and phenotypic characteristics of CD4+ CD25+ Tregs in patients of different hepatitis B virus (HBV) infection status, and investigated the effect of Tregs on antiviral immune responses in CHB patients, and the mechanism of this effect. A total of 137 subjects, including 79 CHB patients, 26 asymptomatic HBV carriers (ASCs), 12 acute hepatitis B (AHB) patients and 20 healthy controls, were enrolled in the study. We found that the frequency of CD4+ CD25(high) Tregs in AHB patients was comparable to that in healthy controls, while it was significantly increased in CHB patients. CD4+ CD25+ Tregs produced interleukin (IL)-10 but little or no interferon (IFN)-gamma under anti-CD3 stimulation. In CHB patients, the frequency of CD4+ CD25(high) Tregs positively correlated with serum viral load, and the Tregs were capable of suppressing the proliferation and IFN-gamma production of autologous peripheral blood mononuclear cells (PBMC) mediated by HBV antigen stimulation in vitro. However, combined administration of anti-programmed death-1 (PD-1) and anti-cytotoxic lymphocyte antigen-4 (CTLA-4) monoclonal antibody slightly enhanced the cellular proliferation and significantly increased the IFN-gamma production of PBMC cocultured with Tregs at a ratio of 2:1. Thus, the frequency of circulating CD4+ CD25+ Tregs is increased in patients with CHB, and this may play an important role in viral persistence by modulating virus-specific immune responses.

Figure 1

Frequency of circulating CD4⁺ CD25⁺ regulatory T cells (Tregs) in various subjects. (a) CD4⁺ T cells were separated into CD25^high, CD25^low and CD25^– T-cell subsets, defined by the fluorescence intensity of CD25 obtained using isotypic control antibody. (b) The frequency of CD4⁺ CD25^high and total CD4⁺ CD25⁺ T cells from chronic hepatitis B (CHB) patients. Data are expressed as box plots, in which the horizontal lines represent the 25th, 50th and 75th percentiles of the measured frequencies of Tregs. (c) The percentages of CD4⁺ CD25^high Tregs in various subjects. The horizontal bars indicate the median percentage of Tregs. The individual frequency for each subject included in the analysis is shown. The significance of differences was calculated using the Kruskal–Wallis H test. (d) The percentage of total CD4⁺ CD25⁺ T cells from different groups. The bars represent the mean (± standard deviation) CD4⁺ CD25⁺ T-cell frequency. AHB, acute hepatitis B (early phase); ASC, asymptomatic hepatitis B virus (HBV) carrier; E⁺, HBV envelope antigen (HBeAg) positive; E^–, (HBeAg) negative; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Figure 2

Characteristics of circulating CD4⁺ CD25⁺ regulatory T cells (Tregs) from chronic hepatitis B (CHB) patients. (a) Representative phenotypic profile of CD45RO, CD45RA, cytotoxic lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) on different CD4⁺ subsets from a CHB patient. (b) The mean expression levels of CD45RO, CD45RA, CTLA-4 and PD-1¹ were determined, with gating on the CD4⁺ CD25^high, CD4⁺ CD25^low and CD4⁺CD25^– T-cell subsets, respectively. **P < 0·01. The bars represent mean ± standard deviation. The significance of differences was calculated using the Kruskal–Wallis H test. CMV, cytomegalovirus; HBeAg, hepatitis B virus envelope antigen.

Figure 3

Forkhead family transcription factor 3 (Foxp3) expression in circulating CD4⁺ CD25⁺ regulatory T cells (Tregs). (a) Foxp3 mRNA level in CD4⁺ CD25⁺ T-cell population from chronic hepatitis B (CHB) patients is presented as expression index calculated by taking the cycle threshold (Ct) value ratio of CD4⁺ CD5⁻ T-cell population as 1. (b) Foxp3 mRNA levels in CD4⁺ CD25⁺ T cells from various subjects are presented as the Ct value ratio of Foxp3:β-actin. The bars represent the mean (± standard deviation) Foxp3 relative expression level. (c) The mean percentage of intracellular Foxp3-positive T cells in various CD4⁺ T-cell subsets from CHB patients. The significance of differences was calculated using the Kruskal–Wallis H test. AHB, acute hepatitis B (early phase); ASC, asymptomatic hepatitis B virus (HBV) carrier.

Figure 4

The cytokine profile of circulating CD4⁺ CD25⁺ regulatory T cells (Tregs). Isolated peripheral blood mononuclear cells (PBMC) and CD4⁺ CD25⁺ T cells from chronic hepatitis B (CHB) patients (n = 12) were cultured separately either with anti-CD3 or with hepatitis B virus surface antigen (HBsAg) as described in the ‘Materials and methods’. The interferon (IFN)-γ (a) and interleukin (IL)-10 (b) levels in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Data are represented as mean ± standard deviation.

Figure 5

Inhibitory capability of circulating CD4⁺ CD25⁺ regulatory T cells (Tregs). (a) The cellular proliferation of isolated peripheral blood mononuclear cells (PBMC) and CD4⁺ CD25⁺ T cells from chronic hepatitis B (CHB) patients under anti-CD3 or hepatitis B virus surface antigen (HBsAg) stimulation. (b) The proliferation of PBMC cocultured with Tregs was determined using the ³H-thymidine incorporation assay. (c) The interferon (IFN)-γ level in the coculture supernatant was measured by enzyme-linked immunosorbent assay (ELISA). *P < 0·05, **P < 0·01, compared with the data for the PBMC group. (d, e) The effects of anti-programmed death-1 (PD-1) and anti-cytotoxic lymphocyte antigen-4 (CTLA-4) monoclonal antibodies used alone or together on cellular proliferation and IFN-γ production of PBMC cocultured with Tregs at a ratio of 2 : 1. All data are shown as the mean ± standard deviation for four to six subjects in each group. The significance of differences was calculated using the Kruskal–Wallis H test. P:T, PBMC:CD4⁺ CD25⁺ Tregs.

Figure 6

Association between regulatory T-cell (Treg) frequency and hepatitis B virus (HBV) DNA load in chronic hepatitis B (CHB) patients. (a) In 71 CHB patients tested, the correlation between circulating CD4⁺ CD25^high Treg frequency and serum HBV DNA load was analysed by Spearman correlation analysis. (b) CD4⁺ CD25^high Treg frequency and HBV DNA load in nine CHB patients naïve to antiviral therapy. (c) CD4⁺ CD25^high Treg frequency and HBV DNA load in these nine CHB patients after antiviral therapy. Each bar represents the percentage of CD4⁺ CD25^high Tregs in each patient. Each triangle in the same panel depicts the viraemia level detected in each patient at the time of Treg analysis.