High frequency of circulating HBcAg-specific CD8 T cells in hepatitis B infection: a flow cytometric analysis

Abstract

Viral antigen-specific T cells are important for virus elimination. We studied the hepatitis B virus (HBV)-specific T cell response using flow cytometry. Three phases of HBV infection were studied: Group A, HBeAg (+) chronic hepatitis; Group B, HBeAb (+) HBV carrier after seroconversion; and Group C, HBsAb (+) phase. Peripheral T cells were incubated with recombinant HB core antigen (HBcAg), and intracytoplasmic cytokines were analysed by flow cytometry. HBcAg-specific CD4 and CD8 T cells were identified in all three groups and the number of IFN-gamma-positive T cells was greater than TNF-alpha-positive T cells. The frequency of IFN-gamma-positive CD4 and CD8 T cells was highest in Group C, compared with Groups A and B. No significant difference in the HBcAg-specific T cell response was observed between Group A and Group B. The HBcAg-specific CD8 T cell response was diminished by CD4 depletion, addition of antibody against human leucocyte antigen (HLA) class I, class II or CD40L. Cytokine-positive CD8 T cells without HBcAg stimulation were present at a high frequency (7 of 13 cases) in Group B, but were rare in other groups. HBcAg-specific T cells can be detected at high frequency by a sensitive flow cytometric analysis, and these cells are important for controlling HBV replication.

Fig. 1

Addition of IL-2 and anti-CD28 to the medium allows a higher frequency of antigen-specific response without increasing the background level. The upper panels show CD4 T cells and the lower panels show CD8 T cells. (a) and (d): Background with IL-2 and anti-CD28 antibody. (b) and (e): Antigen stimulation without IL-2 and anti-CD28 antibody. (c) and (f): antigen stimulation with IL-2 and anti-CD28 antibody.

Fig. 2

Dot plot profile of CD8 T cells for one representative case from Group C: (a) an isotype control; (b) CD8 T cells without HBcAg; (c) CD8 T cells with HBcAg; and (d) CD8 T cells with SEB stimulation. (c) Note the strong response (15·18%) following stimulation with HBcAg.

Fig. 3

Frequency of cytokine-positive subjects in the three groups: (a) without stimulation of HBcAg and (b) with HBcAg stimulation. (a) HBcAg non-specific IFN-γ- and TNF-α-positive CD8 T cells were observed at a high frequency in Group B. (b) HBcAg-specific CD4 or CD8 T cells were observed in all groups. □ normal control; Group A; Group B; ▪ Group C.

Fig. 4

Frequency of HBcAg-specific CD4 and CD8 T cells. A significantly higher frequency was noted for IFN-γ-positive CD4 and CD8, and TNF-α-positive CD8 T cells in Group C.

Fig. 5

Effect of CD4 T cell depletion on cytokine production of HBcAg-specific CD8 T cells for one representative case from Group C. (a) Isotype control, (b) and (d) CD4 and CD8 T cells, respectively, without HBcAg, (c) and (e) CD4 and CD8 T cells, respectively, with HBcAg and (f) HBcAg-stimulated CD8 T cells with CD4 T cell depletion. (f) Note diminution of IFN-γ-positive CD8 T cells following depletion of CD4 T cells.

Fig. 6

Effect of addition of an anti-HLA class I antibody on HBcAg-specific cytokine production in CD4 (upper panels) and CD8 T cells (lower panels) in one representative case from Group C. (a) Isotype control, (b) and (d) CD4 and CD8 T cells, respectively, without HBcAg, (c) and (e) CD4 and CD8 T cells, respectively, with HBcAg and (f) HBcAg-stimulated CD8 T cells plus anti-HLA class I antibody. (f) Note the reduction in HBcAg-specific IFN-γ-positive CD8 T cells in the presence of the anti-HLA class I antibody.

Fig. 7

Addition of anti-HLA class II antibody or anti-CD40L antibody blocks the HBcAg-specific response in both CD4 (upper panels) and CD8 (lower panels) T cells. (a) and (d) CD4 and CD8 T cells, respectively, with HBcAg (b) and (e) CD4 and CD8 T cells, respectively, with HBcAg in the presence of an anti-HLA class II antibody and (c) and (f) CD4 and CD8 T cells, respectively, with HBcAg in the presence of an anti-CD40L antibody.