The level of viral antigen presented by hepatocytes influences CD8 T-cell function

Abstract

CD8 T cells exert their antiviral function through cytokines and lysis of infected cells. Because hepatocytes are susceptible to noncytolytic mechanisms of viral clearance, CD8 T-cell antiviral efficiency against hepatotropic viruses has been linked to their capacity to produce gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). On the other hand, intrahepatic cytokine production triggers the recruitment of mononuclear cells, which sustain acute and chronic liver damage. Using virus-specific CD8 T cells and human hepatocytes, we analyzed the modulation of virus-specific CD8 T-cell function after recognition peptide-pulsed or virally infected hepatocytes. We observed that hepatocyte antigen presentation was generally inefficient, and the quantity of viral antigen strongly influenced CD8 T-cell antiviral function. High levels of hepatitis B virus production induced robust IFN-gamma and TNF-alpha production in virus-specific CD8 T cells, while limiting amounts of viral antigen, both in hepatocyte-like cells and naturally infected human hepatocytes, preferentially stimulated CD8 T-cell degranulation. Our data document a mechanism where virus-specific CD8 T-cell function is influenced by the quantity of virus produced within hepatocytes.

FIG. 1.

Phenotypic analysis of professional antigen-presenting cells and hepatocytes. (A) Monocytes; (B) EBV B cells; (C and D) primary human hepatocytes; (E) HepG2 cells; (F) Huh-7 cells; (G) MG-63 cells; (H) RH1 cells. Cells were stained for primary receptors involved in antigen presentation. All MFI values expressed have the isotype value subtracted. Each panel is representative of at least two separate experiments.

FIG. 2.

Primary hepatocytes can stimulate CD8 T-cell IFN-γ production and degranulation. (A to D) HBc_18-27 T-cell IFN-γ production (A) and HBc_18-27 T-cell degranulation (B) or HBp_455-63 T-cell IFN-γ production (C) and HBp_455-63 T-cell degranulation (D) after 5 h of stimulation by EBV B cells, monocytes, HepG2 cells, and PHH pulsed with increasing concentrations of HBc_18-27 or HBp_455-63 peptide. Data are displayed as the frequency of IFN-γ- or CD107a-positive T-cell clones at each peptide concentration. (E and F) T-cell clone response to target cells pulsed with 10⁻⁸ M peptide. IFN-γ production and degranulation were measured in parallel. The percentages of IFN-γ-positive T cells were plotted against the percentage of CD107a-positive T cells for each target cell line. Each panel is representative of at least three separate experiments. (G) Activation of CMV-specific short-term line. EBV B cells and HepG2 cells were pulsed with 1 μM CMV peptide, and CD107a and IFN-γ production levels were measured after 5 h. (H) Percentage of CMV-specific T-cell activation. CD107a⁺ T cells stimulated by EBV B cells were set at 100% CMV-specific activation. Results for remaining conditions were divided by this number to obtain the percentage of CMV-specific activation under different conditions.

FIG. 3.

Hepatocytes bias the CD8 T-cell response towards degranulation. (A and B) Staining for IFN-γ and CD107a was performed on HBc_18-27 (A) and HBp_455-63 (B) T-cell clones after 5 h of incubation with peptide-pulsed target cells (10⁻⁸ M HBc_18-27 and 5 × 10⁻⁸ M HBp_455-63). Percentages displayed in the upper right quadrant indicate the percent CD107a-positive CD8 T cells that were positive for IFN-γ. Each panel is representative of at least two separate experiments. (C and D) Cytotoxic function of HBc_18-27-specific (C) and HBp_455-63-specific (D) CTL clones. CFSE-labeled HepG2 cells pulsed with either HBc_18-27 (10⁻⁸ M) or HBp_455-63 (5 × 10⁻⁸ M) peptide were mixed with unpulsed HepG2 cells and cultured in the absence or presence of CTL clones (effector/target ratio, 1:1) for 5 h. Cytotoxicity was determined by the disappearance of CFSE-labeled targets.

FIG. 4.

Reduced T-cell cytokine production stimulated by hepatocytes extends to TNF-α and IL-2 and is not altered by the duration of T-cell activation. (A) HBc_18-27 T-cell CD107a and TNF-α production measured by double staining after 5 h of incubation with EBV B cells or HepG2 cells pulsed with the indicated peptide concentrations. (B) IFN-γ, TNF-α, and IL-2 measured in the supernatant of HBc_18-27 T-cell clones cocultured with EBV B cells or HepG2 cells for 5 or 24 h. Data presented are representative of at least two individual experiments.

FIG. 5.

Increased MHC-I expression does not shift the balance in T-cell activation. (A) Phenotype of HepG2 cells with or without 100 U/ml IFN-γ for 24 h. (B) HBc_18-27 and HBp_455-63 T-cell IFN-γ production and degranulation in response to HepG2 cells pulsed with 10⁻⁸ M peptide after 24 h of incubation with IFN-γ.

FIG. 6.

Viral replication within hepatocytes governs the quality of CD8 T-cell responses. (A) HBeAg-HBcAg production with different concentrations of doxycycline. (B) HBc_18-27 (top) or HBp_455-63 (bottom) T-cell clones incubated with the HepG2 cell line containing only tTA. (C) HBc_18-27 (top row) or HBp_455-63 (bottom row) T-cell clone activation after coculture with HepG2.105 cells expressing different levels of HBV. Panels are representative of three separate experiments.

FIG. 7.

HBc_18-27 T-cell recognition of naturally infected hepatocytes. (A) Phenotypes of chronically HBV infected hepatocytes from two individual patients. (B) HBc_18-27-specific T-cell clone degranulation and IFN-γ production after incubation alone with HLA-A2⁺ uninfected, with HLA-mismatched HBV infected, or with HLA-A2⁺ HBV-infected PHH. (C) Graphical representation of CD107a and IFN-γ data from panel B (Exp 1) and an additional experiment with HLA-A2⁺, chronically HBV-infected PHH (Exp 2). A2+ and A2− on the x axis indicate whether PHH were used for the experiment.