Virus-specific CD8+ lymphocytes share the same effector-memory phenotype but exhibit functional differences in acute hepatitis B and C

Abstract

Hepatitis B and hepatitis C viruses (HBV and HCV) are both noncytopathic and can cause acute and chronic infections of the liver. Although they share tropism for the same organ, development of chronic hepatitis is much more frequent following HCV infection, suggesting different mechanisms of viral persistence. In this study, we show that circulating HBV- and HCV-specific tetramer-positive CD8 cells during the acute phase of hepatitis B and C belong almost entirely to an effector-memory subset (CCR7(-) CD45RA(-)). Despite this phenotypic similarity, HBV- and HCV-specific CD8 cells show striking functional differences. HBV-specific tetramer-positive CD8 cells express high perforin content ex vivo, expand vigorously, and display efficient cytotoxic activity and gamma interferon (IFN-gamma) production upon peptide stimulation. A comparable degree of functional efficiency is maintained after the resolution of hepatitis B. In contrast, HCV-specific CD8 cells in the acute phase of hepatitis C express significantly lower levels of perforin molecules ex vivo and show depressed CD8 function in terms of proliferation, lytic activity, and IFN-gamma production, irrespective of the final outcome of the disease. This defect is transient, because HCV-specific CD8 cells can progressively improve their function in patients with self-limited hepatitis C, while the CD8 function remains persistently depressed in subjects with a chronic evolution.

FIG. 1.

Phenotypic analysis of HBV- and HCV-specific CTL. (A) Representative dot plot analyses of HBV and HCV tetramer-positive cells stained with anti-CD8, anti-CCR7, anti-CD27, and anti-CD45RA monoclonal antibodies. (B) Percentages of CCR7⁻ (top) and CD45RA⁻ CCR7⁻ (bottom) cells among HBV tetramer-positive CD8 lymphocytes specific for core 18-27, envelope 335-343, and polymerase 575-583 epitopes and among HCV tetramer-positive CD8 cells specific for NS3 1073-1081, NS3 1406-1415, and NS4B 1992-2000 epitopes in each patient (Pt) with acute HBV and HCV infection. (C) Percentages of CD27⁺ cells among CCR7⁻ CD45RA⁻ tetramer-positive CD8 cells in each patient with acute HBV and HCV infection. (∗), frequency of tetramer-positive cells was <0.01% of CD8⁺ cells; NT, not tested.

FIG. 2.

Peripheral-blood distribution of CD8 differentiation phenotypes during and after the acute stage of hepatitis B and C. The surface expression of CD8, CD45RA, and CCR7 was tested at three sequential time points as indicated in Tables 1 and 2. Each symbol represents the percentage of tetramer-positive cells expressing the indicated phenotypic markers.

FIG. 3.

Representative dot plot analyses of HBV core 18-27 and HCV NS4B 1992-2000 tetramer-positive cells stained with anti-HLA-DR, anti-CCR5, anti-CD45RO, and anti-CD28 monoclonal antibodies. Pt, patient.

FIG. 4.

Analysis of the perforin contents of HBV- and HCV-specific CD8 cells ex vivo. (A) Dot plot analysis of perforin expression by HBV- and HCV-specific CD8 cells stained with anti-perforin monoclonal antibodies. Pt, patient. (B) Percentages of perforin-positive cells assessed ex vivo among the overall population of HBV- or HCV-specific CD8 lymphocytes. PBMC from patients with acute HBV and HCV infection were tested with HBV core 18-27, envelope (env) 335-343, and polymerase (pol) 575-583 and with HCV NS3 1073-1081, NS3 1406-1415, and NS4B 1992-2000 tetramers, respectively. The outcome of the disease (self-limited or chronic evolution) is indicated. ∗, frequency of tetramer-positive cells was <0.01% of CD8⁺ cells; nt, not tested. The percentages of perforin-positive cells in the two groups of patients were significantly different by Student's t test.

FIG. 5.

(A) Capacity for expansion after 10 days of peptide stimulation of HBV and HCV tetramer-positive cells. PBMC from patients with acute HBV and HCV infection were stimulated with HBV core 18-27, envelope (ENV) 335-343, and polymerase (POL) 575-583 and with HCV NS3 1073-1081, NS3 1406-1415, and NS4B 1992-2000 peptides, respectively. Each bar represents the expansion after 10 days of stimulation. The capacity for expansion in the two groups were significantly different by Student's t test. (B) Analysis of the perforin contents of HBV- and HCV-specific CD8 cells from short-term polyclonal CTL lines. Each bar represents the percentage of perforin-positive cells after 10 days of peptide stimulation among the overall population of HBV- or HCV-specific CD8 lymphocytes. (C and D) Functional characterization of HBV- and HCV-specific short-term CTL lines assessed as IFN-γ production (C) and cytolytic activity (D). Virus-specific CD8 cells were analyzed after 10 days of peptide stimulation in patients with acute HBV and HCV infection. ∗, frequency of tetramer-positive cells was <0.01% of CD8⁺ cells; nt, not tested.

FIG.6.

Follow-up analysis of HBV- and HCV-specific CD8 cell frequency and function. The analyses were performed at two different time points (6 to 16 weeks and 24 to 30 weeks after clinical presentation). (A and B) Frequencies and perforin contents of tetramer-positive HBV- and HCV-specific CD8 cells ex vivo. (C, D, E, and F) Capacities for expansion, perforin expression, IFN-γ production, and cytolytic activity after 10 days of stimulation with peptide. (C) Each bar represents the percentage of tetramer-positive cells calculated based on the total number of CD8 cells. (D and E) Each bar represents the percentage of perforin-positive and IFN-γ positive cells, respectively, among the overall population of HBV or HCV tetramer-positive CD8 lymphocytes. (F) The bars represent the percentages of specific lysis expressed by CD8 cells against peptide-pulsed target cells. ∗, frequency of tetramer-positive cells was <0.01% of CD8⁺ cells; nt, not tested.