Volume 137, Issue 4, Pages 1289-1300 (October 2009) Temporal Analysis of Early Immune Responses in Patients With Acute Hepatitis B Virus Infection  Claire Dunn, Dimitra Peppa, Pooja Khanna, Gaia Nebbia, Meleri Jones, Nathan Brendish, R. Monica Lascar, David Brown, Richard J. Gilson, Richard J. Tedder, Geoffrey M. Dusheiko, Michael Jacobs, Paul Klenerman, Mala K. Maini  Gastroenterology  Volume 137, Issue 4, Pages 1289-1300 (October 2009) DOI: 10.1053/j.gastro.2009.06.054 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 The production of innate cytokines is impaired during early acute HBV infection. (A) Circulating concentrations of IFN-α detected in longitudinal serum samples from 3 representative patients assayed by high-sensitivity ELISA. IFN-α concentrations are plotted against liver inflammation (ALT) and viral load (HBV DNA). Changes in 3 major serologic markers of acute HBV infection (anti-HBc IgM, HBeAg, and HBsAg) are depicted as bars above the main graph. Cross-sectional comparison of circulating IFN-α levels from healthy controls, patients with acute HBV at the time of peak viremia (DNA Hi) and at the time of resolution of infection (resolving), and patients with acute hepatitis A virus infection (HAV). Circulating concentrations of (B) IL-15 and (C) IFN-λ1 in sera from patients with acute HBV, patients with acute HAV, and healthy controls. Temporal correlation of HBV viral load and cytokine concentrations detected in longitudinal samples of 3 representative patients with acute HBV. Cross-sectional comparison of healthy donors, patients with acute HBV at the times of peak viral load and resolution, and patients with acute HAV. Any P values of less than .05 are shown. Gastroenterology 2009 137, 1289-1300DOI: (10.1053/j.gastro.2009.06.054) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 The NK cell response is delayed during early acute HBV infection. (A) PBMC samples were stained ex vivo for CD3, CD56, and the activation marker CD69, and the proportion of activated CD3−CD56+ NK cells was identified by flow cytometry. CD69+ NK cells are presented as a percentage of total NK cells. Temporal correlation of NK cell activation, HBV viral load, and ALT level for 3 representative patients. Cross-sectional comparison of ex vivo NK cell activation of healthy controls and patients with acute HBV at time of peak viral load (DNA Hi) and at time of resolution (Resolving). (B) PBMCs were stimulated in vitro with IL-12 and IL-18 and NK cell IFN-γ production determined by intracellular cytokine staining. Temporal correlation of NK cell IFN-γ production, HBV DNA levels, and ALT levels for 3 representative patients. Cross-sectional comparison of IFN-γ production by NK cells from healthy controls and patients with acute HBV at time of peak viral load (DNA Hi) and at the time of resolution (Resolving). Temporal and cross-sectional analysis as in B of (C) NK cell TNF-α production and (D) CD107 expression following stimulation protocols described in Patients and Methods. (E) The percent of NK cells expressing TRAIL and (F) the percent of CD56bright NK cells were calculated directly ex vivo and plotted according to viral load kinetics and for cross-sectional comparison. Gastroenterology 2009 137, 1289-1300DOI: (10.1053/j.gastro.2009.06.054) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Induction of IL-10 in acute HBV. (A) Temporal correlation of circulating concentrations of IL-10 (assayed by sandwich ELISA), liver inflammation (ALT), and viral load (HBV DNA) in longitudinal samples from 3 representative patients. Changes in HBeAg and HBsAg are shown as bars at the top of the graphs. Cross-sectional comparison of circulating IL-10 concentrations obtained for healthy controls and patients with acute HBV at time of peak viremia (DNA Hi) and at time of resolution (Resolving). (B) Temporal correlation of circulating concentrations of IL-10, ALT, and HBV DNA in longitudinal samples from 2 patients with asymptomatic acute HBV infection. Changes in HBeAg, HBsAg, and anti-HBc antibodies are shown as bars at the top of the graphs. (C) Temporal correlation of circulating concentrations of IL-10, IFN-γ production by NK cells, and viral load (HBV DNA) in longitudinal samples from 3 representative patients. (D) PBMCs from patients with acute HBV were stimulated overnight with IL-12 and IL-18 (500 ng/mL each), with or without IL-10 (50 ng/mL) or anti–IL-10 (5 mg/mL) and anti–IL-10 receptor (10 mg/mL) blocking antibodies. NK cell–derived IFN-γ was determined by intracellular cytokine staining. NK cells were identified as CD3−CD56+ by flow cytometry. Shown are 3 representative dot plots taken from one patient and a summary bar chart. Gastroenterology 2009 137, 1289-1300DOI: (10.1053/j.gastro.2009.06.054) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Correlation of HBV-specific CD4 and CD8 T-cell responses with IL-10 levels in early acute HBV. (A) Correlation of IL-10 with ex vivo CD8 T-cell responses quantitated with HLA-A2 peptide tetramers. Tetramer-staining responses for 2 patients against core 18-27 (left panels) and pol 575-83 (right panels)20 were plotted against viral load and IL-10. (B) Correlation of IL-10 with ex vivo functional CD8 T-cell responses against overlapping peptides spanning the HBV genome. (Upper panel) Total CD8 responses were calculated by summing IFN-γ–positive responses to each of the 8 pools of overlapping peptides spanning the HBV genome and plotted (as responses per 500 CD8 T cells) against IL-10 and HBV DNA. *ND, not done because of insufficient PBMCs. (Lower panel) IFN-γ–positive responses were calculated according to HBV protein specificity by summing responses for pools covering each protein and plotted (as responses per 1000 CD8 T cells) against IL-10 and HBV DNA. (C) Correlation of IL-10 with ex vivo IFN-γ–positive CD4 T-cell responses against overlapping peptides spanning the HBV genome. (Upper panel) Total responses and (lower panel) breakdown according to HBV protein specificity, both calculated and plotted as for B. Gastroenterology 2009 137, 1289-1300DOI: (10.1053/j.gastro.2009.06.054) Copyright © 2009 AGA Institute Terms and Conditions