Modulation of Hepatitis C Virus-Specific CD8 Effector T-Cell Function with Antiviral Effect in Infectious Hepatitis C Virus Coculture Model

Abstract

The antiviral effects of hepatitis C virus (HCV)-specific CD8 T cells have been shown in an HCV replicon system but not in an authentic infectious HCV cell culture (HCVcc) system. Here, we developed tools to examine the antigenicity of HCV-infected HLA-A2-positive Huh7.5 hepatoma cells (Huh7.5A2 cells) in activating HCV-specific CD8 T cells and the downstream antiviral effects. Infectious HCV epitope mutants encoding the well-defined genotype 1a-derived HLA-A2-restricted HCV NS3-1073 or NS5-2594 epitope were generated from a genotype 2a-derived HCV clone (Jc1Gluc2A) by site-directed mutagenesis. CD8 T-cell lines specific for NS3-1073 and NS5-2594 were expanded from HCV-seropositive persons by peptide stimulation or engineered from HCV-seronegative donor T cells by transduction of a lentiviral vector expressing HCV-specific T-cell receptors. HCV-specific CD8 T cells were cocultured with Huh7.5 cells that were pulsed with titrating doses of HCV epitope peptides or infected with HCV epitope mutants. HCV-specific CD8 T-cell activation (CD107a, gamma interferon, macrophage inflammatory protein 1β, tumor necrosis factor alpha) was dependent on the peptide concentrations and the relative percentages of HCV-infected Huh7.5A2 cells. HCV-infected Huh7.5A2 cells activated HCV-specific CD8 T cells at levels comparable to those achieved with 0.1 to 2 μM pulsed peptides, providing a novel estimate of the level at which endogenously processed HCV epitopes are presented on HCV-infected cells. While HCV-specific CD8 T-cell activation with cytolytic and antiviral effects was blunted by PD-L1 expression on HCV-infected Huh7.5A2 cells, resulting in the improved viability of Huh7.5A2 cells, PD-1 blockade reversed this effect, producing enhanced cytolytic elimination of HCV-infected Huh7.5A2 cells. Our findings, obtained using an infectious HCVcc system, show that the HCV-specific CD8 T-cell function is modulated by antigen expression levels, the percentage of HCV-infected cells, and the PD-1/PD-L1 pathways and has antiviral and cytotoxic effects. We developed several novel molecular and immunological tools to study the interactions among HCV, HCV-infected hepatocytes, and HCV-specific CD8 T cells. Using these tools, we show the level at which HCV-infected hepatoma cells present endogenously processed HCV epitopes to HCV-specific CD8 T cells with antiviral and cytotoxic effects. We also show the marked protective effect of PD-L1 expression on HCV-infected hepatoma cells against HCV-specific CD8 T cells.

Pathogenetic Mechanisms of T Cell Dysfunction in Chronic HBV Infection and Related Therapeutic Approaches

“…In vitro studies have addressed the interplay between infected hepatocytes and anti-viral T cells, showing the strengthening of the T cell function as antigen expression increases, suggesting that high viral antigen production is needed for efficient T-cell activation within the liver (67). On the same line, an antigen dose-dependent anti-viral T cell function increment has been observed also in an infectious HCV-hepatoma cell co-culture model, in which a cognate epitope expression threshold has been investigated, indicating a peptide concentration range for effector T cell activation (68). Also the effector:target ratio has been shown to modulate the HCV-specific T cell function in an HCV replicon system, with non-cytolytic mechanisms prevailing at lower ratio values (69).…”

“…Also the effector:target ratio has been shown to modulate the HCV-specific T cell function in an HCV replicon system, with non-cytolytic mechanisms prevailing at lower ratio values (69). However, as in vivo high antigen loads are associated to co-inhibitory receptor/ligand overexpression, when PD-L1 expressing hepatoma cells were used, recapitulating more closely the liver environment, the T cell cytolytic activity resulted significantly inhibited (68). Indeed, experiments performed in animal models, evidenced a more complex T cell regulation in the liver environment.…”

Dissection of two drug-targeted regions of Hepatitis C virus subtype 4a infecting Egyptian patients

“…Transduction and engineering of CD4+ and CD8+ T cells against different HCV epitopes (e.g. NS3 1073-1081, NS5A 1992-2000 and NS5B 2594-2602) via T cell receptor (TCR) gene transfer confer antiviral and cytotoxic activity and produce cytokines (IFN-γ, IL-2 and TNF-α) [101,102]. Zhang et al [103] engineered high affinity TCRs targets (HCV NS3 1406-1415 epitope) and constructed a molecule called high-affinity T-cell activation core (HATac) through the fusion of high affinity TCRs with a T-cell activation molecule (anti-CD3 single-chain variable fragment) and this molecule succeeded to redirect functional non-specific CD8 T cells to recognize and kill cells presenting HCV NS3 antigens.…”

Humanized Mice as an Effective Evaluation System for Peptide Vaccines and Immune Checkpoint Inhibitors

“…Viral infections do not always enhance PD-L1 expression, because similar PD-L1 levels are detected in individuals not infected with viruses [61][62][63][64]. Increased PD-L1 levels are related to specific viruses, such as the following: Epstein-Barr virus (EBV) [65][66][67][68], hepatitis B virus (HBV) [69][70][71], hepatitis C virus (HCV) [72][73][74][75], human immunodeficiency virus (HIV) [63,[76][77][78][79], human papilloma virus (HPV) [68,[80][81][82][83], Merkel cell polyomavirus (MCPyV) [84], bovine leukemia virus (BLV) [85], and Kaposi sarcoma-associated herpes virus (KSHV) [86]. The pathobiological mechanisms by which viruses trigger the expression of PD-L1 have been elucidated.…”