title: 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 stimulationin vitroor 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.IMPORTANCEWe 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.
authors.affiliation: Corporal Michael J. Crescenz Veterans Affairs Medical Center, Philadelphia, Pennsylvania, USA.|University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.|Division of Gastroenterology, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ojiro
authors.affiliation: Corporal Michael J. Crescenz Veterans Affairs Medical Center, Philadelphia, Pennsylvania, USA.|University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Xiaowang
authors.initials: X
authors.lastname: Qu
authors.affiliation: Corporal Michael J. Crescenz Veterans Affairs Medical Center, Philadelphia, Pennsylvania, USA.|University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Hyosun
authors.initials: H
authors.lastname: Cho
authors.affiliation: Corporal Michael J. Crescenz Veterans Affairs Medical Center, Philadelphia, Pennsylvania, USA.|University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Jang-June
authors.initials: JJ
authors.lastname: Park
authors.affiliation: Immunocore Ltd., Abingdon, Oxfordshire, United Kingdom.
authors.forename: Annelise
authors.initials: A
authors.lastname: Vuidepot
authors.affiliation: Immunocore Ltd., Abingdon, Oxfordshire, United Kingdom.
authors.forename: Nikolai
authors.initials: N
authors.lastname: Lissin
authors.affiliation: Immunocore Ltd., Abingdon, Oxfordshire, United Kingdom.
authors.forename: Peter E
authors.initials: PE
authors.lastname: Molloy
authors.affiliation: Adaptimmune Ltd., Abingdon, Oxfordshire, United Kingdom.
authors.forename: Alan
authors.initials: A
authors.lastname: Bennett
authors.affiliation: Immunocore Ltd., Abingdon, Oxfordshire, United Kingdom.
authors.forename: Bent K
authors.initials: BK
authors.lastname: Jakobsen
authors.affiliation: Corporal Michael J. Crescenz Veterans Affairs Medical Center, Philadelphia, Pennsylvania, USA.|University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: David E
authors.initials: DE
authors.lastname: Kaplan
authors.affiliation: University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: James L
authors.initials: JL
authors.lastname: Riley
authors.affiliation: Corporal Michael J. Crescenz Veterans Affairs Medical Center, Philadelphia, Pennsylvania, USA kmchang@mail.med.upenn.edu.|University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Kyong-Mi
authors.initials: KM
authors.lastname: Chang
mesh_terms: D060890:B7-H1 Antigen / Q000235:genetics; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D045744:Cell Line, Tumor; D054407:Chemokine CCL4 / Q000235:genetics; D018920:Coculture Techniques; D003601:Cytotoxicity Tests, Immunologic; D015789:HLA-A2 Antigen / Q000276:immunology; D016174:Hepacivirus / Q000235:genetics / Q000276:immunology*; D022781:Hepatocytes / Q000276:immunology / Q000821:virology*; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics; D008213:Lymphocyte Activation; D052118:Lysosomal-Associated Membrane Protein 1 / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000494:pharmacology; D011948:Receptors, Antigen, T-Cell / Q000235:genetics; D014161:Transduction, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
chemical_list: D060890:B7-H1 Antigen; C423236:CD274 protein, human; D054407:Chemokine CCL4; D015789:HLA-A2 Antigen; D052118:Lysosomal-Associated Membrane Protein 1; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma
keywords: CD8 T cells; HCV-specific TCR; Huh7.5 cells; PD-1 blockade; coculture; lentiviral transduction