Volume 138, Issue 5, Pages 1885-1897.e10 (May 2010) Interferon-α–Induced TRAIL on Natural Killer Cells Is Associated With Control of Hepatitis C Virus Infection  Kerstin A. Stegmann, Niklas K. Björkström, Heike Veber, Sandra Ciesek, Peggy Riese, Johannes Wiegand, Johannes Hadem, Pothakamuri V. Suneetha, Jerzy Jaroszewicz, Chun Wang, Verena Schlaphoff, Paraskevi Fytili, Markus Cornberg, Michael P. Manns, Robert Geffers, Thomas Pietschmann, Carlos A. Guzmán, Hans–Gustaf Ljunggren, Heiner Wedemeyer  Gastroenterology  Volume 138, Issue 5, Pages 1885-1897.e10 (May 2010) DOI: 10.1053/j.gastro.2010.01.051 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 IFN-alfa stimulation up-regulates TRAIL mRNA in NK cells. (A) Signal log ratios of 6-hour medium vs ex vivo against 6-hour medium vs 6-hour stimulation with 100 ng/mL IFN-alfa-2b and (B) signal log ratios of 6-hour stimulation with IFN-alfa-2b 100 ng/mL vs 6-hour medium against 6-hour stimulation with IFN-alfa-2b 1 ng/mL vs 6-hour medium for RNA isolated from purified NK cells pooled from 5 healthy controls. Arrows denote TNFSF10 mRNA (grey rhombs). (C) Up-regulation of TNFSF10 mRNA was confirmed by real-time RT-PCR. RSP9-mRNA was used as control. Fold-increase was normalized to ex vivo sample. Gastroenterology 2010 138, 1885-1897.e10DOI: (10.1053/j.gastro.2010.01.051) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 IFN-alfa, but not ribavirin, up-regulates TRAIL on the surface of NK cells. (A) TRAIL expression on CD56bright and CD56dim NK cells from one representative healthy donor after IFN-alfa-2b stimulation of PBMCs. Gating on lymphocytes in forward scatter vs side scatter was performed. (B) TRAIL expression at different time points on CD56bright (upper panel) and CD56dim NK cells (lower panel) after stimulation with 100 ng/mL (triangles) or 1 ng/mL (rhomb) of IFN-alfa-2b, PEG–IFN-alfa-2a, and PEG–IFN-alfa-2b. One representative individual of 6 is shown. (C) TRAIL expression on CD56bright (upper panel) and CD56dim NK cells (lower panel) after 6-hour stimulation with IFN-alfa-2b, PEG–IFN-alfa-2a, and PEG–IFN-alfa-2b. Two representative individuals of 6 are shown. (D) TRAIL expression on PBMCs stimulated with different concentrations of ribavirin. Two representative individuals of 6 are shown. Gastroenterology 2010 138, 1885-1897.e10DOI: (10.1053/j.gastro.2010.01.051) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Low ex vivo levels of TRAIL detected on NK cells from patients. Individual data of TRAIL expression level (mean fluorescence intensity [mfi]) and frequency of TRAIL-positive CD56bright (left) and CD56dim NK cells (right) of healthy controls (n = 11, filled rhombs), HCV-recovered (n = 5, open rhombs), chronic hepatitis C (n = 13, triangles), and chronic hepatitis B patients (n = 10, circles) is shown. *P < .05. Gastroenterology 2010 138, 1885-1897.e10DOI: (10.1053/j.gastro.2010.01.051) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 NK cells from HCV-recovered patients are superior at up-regulating TRAIL after in vitro IFN-alfa-2b stimulation. Individual data of TRAIL expression level (mean fluorescence intensity [mfi]) and frequency of TRAIL-positive CD56bright (left) and CD56dim NK cells (right) after 6-hour stimulation with 1 and 100 ng/mL IFN-alfa-2b of healthy controls (n = 11, filled rhombs), HCV-recovered (n = 5, open rhombs), chronic hepatitis C (n = 13, triangles), and chronic hepatitis B patients (n = 10, circles) is shown. *P < .05. Gastroenterology 2010 138, 1885-1897.e10DOI: (10.1053/j.gastro.2010.01.051) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 In vivo TRAIL expression increase on NK cells after IFN-alfa administration to patients. (A) Mean TRAIL expression (mean fluorescence intensity [mfi]) and frequency of TRAIL-positive CD56bright (left) and CD56dim (right) NK cells from healthy controls (n = 10) and acute hepatitis C patients (n = 12) at different time points during monotherapy with PEG–IFN-alfa-2b. *P < .05. (B) Serum ALT (dotted line) and serum HCV-RNA levels (black line) as well as TRAIL MFI on CD56bright (white rhombs) and CD56dim NK cells (grey rhombs) from 5 acute hepatitis C patients during treatment. Grey bars illustrate during which periods of time the patients received treatment. (C) TRAIL expression on CD56bright (white) and CD56dim NK cells (grey) and serum HCV-RNA level (black) right before and 2 days after PEG–IFN-alfa-2a treatment shown for 6 chronic hepatitis C patients. (D) Representative staining of TRAIL in one patient from panel C with the frequency of TRAIL expressing CD56bright NK cells indicated. Gastroenterology 2010 138, 1885-1897.e10DOI: (10.1053/j.gastro.2010.01.051) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 IFN-alfa–stimulated NK cells kill Huh7.5 cells through TRAIL. (A) Specific lysis of Jurkat, K562, Huh-7, and Huh7.5 cells by purified resting and IFN-alfa–stimulated NK cells. One representative experiment of 4 is shown. (B) Lysis of Huh7.5 cells by resting or IFN-alfa–stimulated NK cell at an effector to target ratio of 5:1 with or without addition of blocking anti-TRAIL antibody. Mean values for 4 healthy donors are shown. (C) Lysis of Huh7.5 cells by resting or IFN-alfa–stimulated NK cells at an E:T ratio of 5:1 with or without addition of TRAIL-R2/Fc and Fas/Fc-chimeras. Bovine serum albumin (bsa) was used as control. Mean values for 4 healthy donors are shown. (D) Lysis of Huh7.5 cells by resting or IFN-alfa–stimulated NK cells at an E:T ratio of 5:1 with different concentrations of blocking anti-TRAIL antibody. MopC21 was used as control antibody. Mean values of 4 healthy donors are shown. (E) Lysis of Huh7.5 cells by sorted CD56bright and CD56dim NK cells at an E:T ratio of 5:1 with or without addition of blocking anti-TRAIL antibody. Mean values for 6 healthy donors are shown. *P < .05. (F) Lysis of Huh7.5 cells by thawed whole PBMCs of chronic hepatitis C patients at an E:T ratio of 100:1. Samples were obtained of 9 patients before treatment and of 4 patients additionally on day 2/3 of IFN-alfa–based treatment. Gastroenterology 2010 138, 1885-1897.e10DOI: (10.1053/j.gastro.2010.01.051) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 IFN-alfa–stimulated NK cells kill HCV-replicating Huh7.5 cells through TRAIL. In all experiments, close to 100% of Huh7.5 cells were NS5A-positive. Microscopic pictures of representative cell cultures of adherent HCV-RNA–transfected and HCV-RNA–replicating Huh7.5 cells after addition of (A) IFN-alfa-2b–containing media, (B) resting NK cells, (C) IFN-alfa-2b–stimulated NK cells, and (D) IFN-alfa-2b–stimulated NK cells and 10 μg/mL of blocking anti-TRAIL antibody are given. (E) Luciferase assay for HCV-RNA–transfected and replicating Huh7.5 cells incubated without or with IFN-alfa-2b and with or without IFN-alfa-2b–stimulated NK cells and addition of anti-TRAIL or anti-CD13. (F) Flow cytometry-based killing assay with HCV-infected and HCV-uninfected Huh7.5 cells incubated without or with IFN-alfa-2b and with or without IFN-alfa-2b–stimulated NK cells and addition of anti-TRAIL or anti-CD13. One representative experiment of 6 is shown. Gastroenterology 2010 138, 1885-1897.e10DOI: (10.1053/j.gastro.2010.01.051) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 NK cell from HCV-recovered patients are superior at up-regulating TRAIL after in vitro IFN-alfa-2b stimulation. Frequency of TRAIL-positive NK cells after 20 hours of IFN-alfa-2b stimulation. Individual data for HCV-recovered (n = 9; open rhombs) and IFN-alfa–nonresponder patients (n = 16, filled rhombs) are shown. *P < .05. Gastroenterology 2010 138, 1885-1897.e10DOI: (10.1053/j.gastro.2010.01.051) Copyright © 2010 AGA Institute Terms and Conditions