Volume 144, Issue 2, Pages 426-436 (February 2013) Factors That Determine the Antiviral Efficacy of HCV-Specific CD8+ T Cells Ex Vivo  Bianca Seigel, Bertram Bengsch, Volker Lohmann, Ralf Bartenschlager, Hubert E. Blum, Robert Thimme  Gastroenterology  Volume 144, Issue 2, Pages 426-436 (February 2013) DOI: 10.1053/j.gastro.2012.10.047 Copyright © 2013 AGA Institute Terms and Conditions

Figure 1 Differential antiviral efficacy of virus-specific CD8+ T cells. (A) Ex vivo tetramer frequencies from all patients and representative tetramer stainings after CD8+ T-cell selection are shown. Patients with ex vivo tetramer-positive CD8+ T cells ≥0.01% were included. (B) Ex vivo antiviral efficacy of virus-specific CD8+ T cells targeting Flu-, EBV-, CMV-, or HCV-specific epitopes was analyzed. Huh7A2HCV cells were pulsed with the respective viral peptides and cocultured for 72 hours with CD8+ T cells at an E/T ratio of 2:1. Only consensus and not autologous variant peptides were used. Inhibition of replication was measured by luciferase activity. (C–E) Ex vivo antiviral efficacy of virus-specific CD8+ T cells targeting (C) HCV-, (D) EBV-, and (E) Flu-specific epitopes was analyzed. Correlation of tetramer frequencies with inhibition of replication is shown. Each dot represents one patient. cHCV, chronic hepatitis C virus. Gastroenterology 2013 144, 426-436DOI: (10.1053/j.gastro.2012.10.047) Copyright © 2013 AGA Institute Terms and Conditions

Figure 2 Antiviral efficacy is linked to IFN-γ production of virus-specific CD8+ T cells. (A) Comparison of tetramer-positive CD8+ T cells and CD8+ T cells producing IFN-γ after peptide-specific stimulation for 5 hours. (B) Huh7A2HCV cells pulsed with Flu-, EBV-, or HCV-specific viral peptides and cocultured for 72 hours with CD8+ T cells at an E/T ratio of 2:1 and additionally in the presence of 10 μg/mL anti–IFN-γ. (C and D) Representative stainings and antiviral efficacy of one patient. (C) Percent values indicate the tetramer-positive CD8+ T cells after CD8+ T-cell selection (upper panel) and IFN-γ+CD8+ T cells (lower panel), respectively. (D) Huh7A2HCV cells were pulsed with Flu-, EBV-, or HCV (NS31073)-specific viral peptides and cocultured for 72 hours with CD8+ T cells at an E/T ratio of 2:1. Inhibition of replication was measured by luciferase activity. Assay was performed in triplicate. (E and F) Tetramer-positive CD8+ T cells were fluorescence-activated cell sorted and cocultured with 30,000 Huh7A2HCV cells pulsed with Flu-, EBV-, or HCV-specific viral peptides for 72 hours at different E/T ratios. Cell numbers of tetramer-positive CD8+ T cells are indicated. cHCV, chronic hepatitis C virus. Gastroenterology 2013 144, 426-436DOI: (10.1053/j.gastro.2012.10.047) Copyright © 2013 AGA Institute Terms and Conditions

Figure 3 Antiviral efficacy correlates with expression levels of CD127. (A) Two groups of HCV-specific CD8+ T cells can be distinguished by level of CD127 expression. High levels of CD127 expression (>80%) are displayed by black boxes and low expression by black circles. (B) Two representative stains for low CD127 and high CD127 expression levels (gated on CD8+ T cells). (C) Expression of PD-1 and 2B4 is shown for CD127− and CD127+ HCV-specific CD8+ T cells (***P = .0006; *P = .027). (D) Frequencies are shown for CD127− and CD127+ HCV-specific T cells. (E) Inhibition of replication of CD127− and CD127+ HCV-specific CD8+ T cells was measured by luciferase activity (P = .0451). Gastroenterology 2013 144, 426-436DOI: (10.1053/j.gastro.2012.10.047) Copyright © 2013 AGA Institute Terms and Conditions

Figure 4 Antiviral efficacy is inversely correlated to expression levels of PD-1. (A and B) Huh7A2HCV cells were pulsed with HCV-specific viral peptides and cocultured for 72 hours with CD8+ T cells at an E/T ratio of 2:1. Correlation of (A) PD-1 and (B) 2B4 expression and inhibition of replication is shown. Representative tetramer staining for low and high (A) PD-1 and (B) 2B4 expression is shown. Stainings are gated on CD8+ T cells. Gastroenterology 2013 144, 426-436DOI: (10.1053/j.gastro.2012.10.047) Copyright © 2013 AGA Institute Terms and Conditions

Figure 5 Differential cytokine responsiveness of CD127− and CD127+ HCV-specific CD8+ T cells. (A) Huh7A2HCV cells labeled with HCV (NS31406)-specific viral peptide and cocultured for 72 hours with CD8+ T cells of one representative patient at an E/T ratio of 2:1 in the presence of cytokines. (Left panel) Unlabeled Huh7A2HCV cells. Dashed lines indicate the baseline effect of adding CD8+ T cells to Huh7A2HCV cells. Assays were performed independently at 3 different time points. ***P <.001. (B) Huh7A2HCV cells were labeled with viral peptides and cocultured for 72 hours with CD8+ T cells at an E/T ratio of 2:1 in the presence of cytokines or blocking reagents. Open boxes indicate CD127+PD-1− and black boxes indicate CD127−PD-1+ HCV-specific CD8+ T cells. Inhibition of replication was measured by luciferase activity. An increase in inhibition of replication compared with baseline is depicted. A total of 7 chronically infected patients were analyzed. Tetramer-specific responses with >50% PD-1 expression were categorized as PD-1+. Gastroenterology 2013 144, 426-436DOI: (10.1053/j.gastro.2012.10.047) Copyright © 2013 AGA Institute Terms and Conditions

Figure 6 Differential antiviral efficacy of HCV-specific CD8+ T cells of patients with SVR and patients with SpR. (A) Ex vivo antiviral efficacy of virus-specific CD8+ T cells targeting HCV-specific epitopes obtained from patients with chronic HCV infection (cHCV), SVR, and SpR were analyzed. Huh7A2HCV cells were pulsed with the HCV-specific peptides and cocultured for 72 hours with CD8+ T cells at an E/T ratio of 2:1. Inhibition of replication was measured by luciferase activity as relative luciferase units. (B) Comparison of tetramer-positive CD8+ T cells and CD8+ T cells producing IFN-γ after peptide-specific stimulation for 5 hours. (C) The coculture assay was performed additionally in the presence of 10 μg/mL anti–IFN-γ. Results for patients with SpR and patients with cHCV are shown. (D) Expression of CD127, PD-1, and 2B4 on HCV-specific CD8+ T cells of patients with SpR is shown. Gastroenterology 2013 144, 426-436DOI: (10.1053/j.gastro.2012.10.047) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 1 Gastroenterology 2013 144, 426-436DOI: (10.1053/j.gastro.2012.10.047) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 2 Gastroenterology 2013 144, 426-436DOI: (10.1053/j.gastro.2012.10.047) Copyright © 2013 AGA Institute Terms and Conditions