Volume 132, Issue 2, Pages 654-666 (February 2007) Discordant Role of CD4 T-Cell Response Relative to Neutralizing Antibody and CD8 T- Cell Responses in Acute Hepatitis C  David E. Kaplan, Kazushi Sugimoto, Kimberly Newton, Mary E. Valiga, Fusao Ikeda, Ayse Aytaman, Frederick A. Nunes, Michael R. Lucey, Barbara A. Vance, Robert H. Vonderheide, K. Rajender Reddy, Jane A. McKeating, Kyong–Mi Chang  Gastroenterology  Volume 132, Issue 2, Pages 654-666 (February 2007) DOI: 10.1053/j.gastro.2006.11.044 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Clinical and virologic course of patients with acute hepatitis C are shown in line graphs for 5 subjects with acute resolution (AR) and 11 subjects with acute to chronic evolution (AC) with the infecting HCV genotype shown in parenthesis and clinical onset (week 0) defined by the first detected alanine aminotransferase (ALT) elevation. The black line graphs show the serum ALT levels, and red line graphs show HCV RNA titer. For HCV RNA, red diamond indicates positive HCV RNA by quantitative RT-PCR (detection limit 600 IU/mL); blue diamond indicates negative HCV RNA by sensitive Roche COBAS TaqMan HCV or qualitative Amplicore v2.0 with detection limit of 10 and 50 IU/mL, respectively; red plus signs for AR1 and AR2 indicate that serum HCV RNA was detectable by Roche COBAS qualitative Amplicore v2.0 but not quantified by quantitative PCR. The horizontal dotted line indicates the upper limit of normal for serum ALT (63 U/mL). Shaded horizontal bars for the 6 graphs on the far right indicate the timing of IFN-α-based antiviral therapy. Gastroenterology 2007 132, 654-666DOI: (10.1053/j.gastro.2006.11.044) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Inverse correlation between HCV-specific CD4 T-cell responses and HCV RNA in acute hepatitis C. (A) Evolution in HCV-specific CD4 proliferative T-cell response (in stimulation index or SI) is shown as bar graphs for genotype 1-derived HCV core (open), NS3/4 (solid), and NS5 (shaded) in 14 genotype 1-infected patients relative to HCV RNA titer in log10 IU/mL (red line graph). Undetectable HCV RNA below 50 IU/mL (detection limit of qualitative PCR) is depicted as 10 IU/mL. Top 5 graphs show the responses in patients with sustained or transient viral clearance during the first year. Lower 9 graphs show the responses in AC patients without spontaneous viral clearance. Horizontal gray bars represent periods of antiviral therapy. (B) Evolution in HCV-specific CD4 T-cell IFN-γ response is shown as bar graphs for genotype 1-derived HCV core (open), NS3/4 (solid), and NS5 (shaded) in 14 genotype 1-infected patients relative to HCV RNA titer (red line graph). Red asterisks indicate time points without sufficient PBMC to measure HCV-specific IFN-γ response. (C) Significant inverse correlation between HCV-specific CD4 T-cell proliferation and HCV RNA titer. The combined sum of CD4 proliferative T-cell responses to HCV core, NS3/4, and NS5 is compared with respective HCV RNA titer directly (left) or in subgroup analysis based on low or high HCV RNA titer with a cutoff of 1000 IU/mL (right). The data reflect time points within 1 year of clinical onset, excluding those obtained during IFN-α therapy. P values were determined by Generalized Estimating Equation analysis, correcting for varied number of observations per subject. (D) Lack of significant correlation between HCV-specific CD4 T-cell IFN-γ response and HCV RNA titer. The sum of IFN-γ responses to HCV core, NS3/4, and NS5 is compared with respective HCV RNA titer directly (left) or in subgroup analysis based on low or high HCV RNA titer with a cutoff of 1000 IU/mL (right). The data reflect time points within 1 year of clinical onset, excluding those during IFN-α therapy. P values were determined by Generalized Estimating Equation analysis, correcting for varied number of observations per subject. Gastroenterology 2007 132, 654-666DOI: (10.1053/j.gastro.2006.11.044) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Detection of HCV-specific CD8 T cells during acute hepatitis C with chronic evolution and resolution. (A) Flow cytometric analysis of 7 HLA-A2+ patients at the earliest time points using MHC-peptide tetramers shows that HCV-specific CD8 T cells are induced during acute HCV infection regardless of virologic outcome. The FACS density plots show tetramer+CD8+ cells and their respective frequencies per CD8 T cells on the upper right quadrant specific for HCV NS3 1073, HCV NS3 1406, and influenza matrix epitopes. Concurrent ALT activity (U/dL), HCV RNA titers (IU/mL), sum HCV-specific CD4 T-cell proliferation (SI), and IFN-γ (SFU/million PBMC) are shown below. (B) Evolution in HCV-specific CD8 T-cell frequency based on percentage tetramer+/CD8 for NS3 1073 (shaded bars) and NS3 1406 (open bars) is shown for 7 HLA-A2+ patients demonstrating reduced frequency beyond 1–2 years in all 3 AC patients, although similar trend could not be assessed in AR patients followed for <1 year. (C) Evolution of HCV-specific IFN-γ+ CD8 T cells measured ex vivo by IFN-γ Elispot during first year of follow-up is shown for 10 subjects. CD4-depleted PBMC were stimulated with pools of overlapping 15mer HCV peptides spanning core and NS3-5, and the pools were summed to provide total HCV-specific CD8 IFN-γ+ response. Strong CD8 IFN-γ+ response (greater than 1000 SFU/106 PBMC) was detected in 2 of 4 AR and 3 of 6 AC patients. Gastroenterology 2007 132, 654-666DOI: (10.1053/j.gastro.2006.11.044) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 HCV-specific CD8 T cells without concurrent antigen-specific IFN-γ production ex vivo in acute hepatitis C with chronic evolution. (A, B) Detection of HCV NS3 1073-specific CD8 T cells without antigen-specific IFN-γ production ex vivo at weeks 2, 11, 27, and 76 in peripheral blood of patient AC11 is demonstrated by FACS plots with direct HLA-A2-HCV NS3 1073 tetramer staining (A) and intracellular IFN-γ staining with and without 5 hours of NS3 1073 epitope peptide stimulation (B) as previously described.14,16 Frequency of tetramer+ or HCV-specific IFN-γ+ cells per CD8 T cells is shown on right upper quadrant of each density plot, and serum HCV RNA titer (IU/mL) at each time point is indicated below. (C) Expansion of CD8 T cells with efficient NS3 1073-specific effector function in vitro was achieved with biweekly rhIL-2 (20 U/mL) and weekly restimulation with autologous irradiated feeder cells and NS3 1073 peptide (10 μg/mL) as previously described,14 resulting in NS3 1073-specific IFN-γ and TNF-α production by intracellular cytokine staining and NS3 1073-specific cytolysis by standard 4-hour 51chromium release assay as previously described14 at effector:target ratio of 50:1. Gastroenterology 2007 132, 654-666DOI: (10.1053/j.gastro.2006.11.044) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Fluctuations in strength and scope of HCV-specific total and CD8 T-cell IFN-γ responses during acute hepatitis C relative to virologic outcome. (A) Peak HCV-specific total and CD8 T-cell IFN-γ responses in acute hepatitis C within 6 months of clinical onset are shown, combining 12 consecutive regions based on IFN-γ Elispot with overlapping peptide pools derived from the entire HCV core (pool 1, purple bar), NS3 and NS4 (pools 2–6, green bars), and NS5 (pools 7–12, orange/brown bars) in 3 AR and 5 AC patients with genotype 1 infection. Total T-cell IFN-γ responses in whole PBMC are shown in spot-forming units (SFU) per million PBMC with significantly greater frequency (median, 3370 vs 643 SFU/106 PBMC, P = .025) but without a clearly dominant antigenic region associated with outcome. (B) Fluctuating scope and specificity of HCV-specific total T-cell IFN-γ response in acute hepatitis C with chronic evolution. Total T-cell IFN-γ responses to 12 consecutive regions in HCV core (pool 1, purple), NS3/NS4 (pools 2–6, green bars), and NS5 (pools 7–12, orange/brown bars) are shown at multiple time points for 2 AR and 3 AC patients. The assay time points in weeks are shown on upper left corner of each graph The number of immunogenic pools at each time point (based on a cutoff of 47 HCV-specific IFN-γ+ SFU/106 PBMC, which is 3 standard deviations above mean SFU in unstimulated wells) is also shown in parenthesis for each graph. Both AR patients sustain a broad response, whereas all 3 AC patients show fluctuating scope and strength over time. Gastroenterology 2007 132, 654-666DOI: (10.1053/j.gastro.2006.11.044) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Neutralizing antibody (nAb) response in acutely HCV-infected patients with spontaneous resolution or chronic evolution. Cross-reactive nAb responses to HCVpp derived from H77 (genotype 1a, blue lines) and Con1 (genotype 1b, black lines) were measured in 14 genotype 1-infected and 2 nongenotype 1-infected patients. Shaded areas indicate HCV RNA titer. Hatched horizontal red bars indicate periods of IFN-α-based antiviral therapy. Dotted horizontal lines indicate the cutoff of 50% for a significant nAb response as described in the Materials and Methods section. Gastroenterology 2007 132, 654-666DOI: (10.1053/j.gastro.2006.11.044) Copyright © 2007 AGA Institute Terms and Conditions