Volume 134, Issue 7, Pages 1938-1949.e3 (June 2008) Dynamic Programmed Death 1 Expression by Virus-Specific CD8 T Cells Correlates With the Outcome of Acute Hepatitis B  Zheng Zhang, Ji–Yuan Zhang, E. John Wherry, Bo Jin, Bin Xu, Zheng–Sheng Zou, Shu–Ye Zhang, Bao–Sen Li, Hui–Feng Wang, Hao Wu, George K.K. Lau, Yang–Xin Fu, Fu–Sheng Wang  Gastroenterology  Volume 134, Issue 7, Pages 1938-1949.e3 (June 2008) DOI: 10.1053/j.gastro.2008.03.037 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 PD-1 is up-regulated on HBV-specific CD8 T cells early after clinical onset in patients with AHB but not in patients with ALF. (A) Representative dot plots of PD-1 expression on HBV-, CMV-, and Flu-specific CD8 T cells. Values in the upper right quadrant indicate the percentage of PD-1–positive pentamer cells. (B) PD-1 percentage (left) and MFI (right) on HBV-, CMV-, and Flu-specific CD8 T cells in HBV-infected patients and healthy subjects. Each dot represents one subject. (C) Percentage (left) and MFI (right) of PD-1–positive pentamer cells by epitope specificity in patients with AHB and ALF. Horizontal bars indicate the median PD-1 percentage or MFI. (D) Longitudinal detection of PD-1 expression by HBV-specific CD8 T cells in representative patients with AHB and ALF. Values in the upper right quadrant indicate the percentage of pentamer-positive CD8 T cells that express PD-1. Gastroenterology 2008 134, 1938-1949.e3DOI: (10.1053/j.gastro.2008.03.037) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 In situ liver infiltration of PD-1–positive or PD-L1–positive cells is increased in patients with AHB. (A) Immunohistochemical staining for intrahepatic PD-1–positive and PD-L1–positive cells in HBV-infected patients and healthy controls (HC). High-power field, original magnification 400×. (B) Numbers of in situ liver portal and lobular PD-1– or PD-L1–positive cells in patients with AHB (n = 6), patients with CHB (n = 11), and HC subjects (n = 4). (C) Colocalization of PD-1 and PD-L1 with various cell markers is shown by immunofluorescence double staining in liver biopsy specimens from patients with AHB. PD-1 (green) is colocalized with both CD8 and CD4 (red) T cells. PD-L1 (green) is colocalized with CD68-positive macrophages (red), CD31-positive epithelial cells (red), and CD11c-positive dendritic cells (red). The 2-color merged panels were shown with colocalization visible in yellow. Representative staining at an original magnification of 400× is shown. Gastroenterology 2008 134, 1938-1949.e3DOI: (10.1053/j.gastro.2008.03.037) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 PD-1 expression is significantly correlated with liver injury severity during the early phase of acute HBV infection. (A and B) Significant correlations were found between the PD-1 expression (percentage, left; MFI, right) on (A) HBV-specific CD8 T cells or (B) total CD8 T cells and serum ALT levels at week 1–2 after clinical onset in 23 patients with AHB. Solid line, linear growth trend; r, correlative coefficient. P values are shown. (C) Longitudinal PD-1 expression on HBV-, CMV-, and Flu-specific CD8 T cells from 4 representative patients with AHB. The percentage of PD-1–positive pentamer cells (bars) is indicated on the left y-axis and the serum ALT level (black line) on the right y-axis. The plasma HBV DNA loads (copies/mL) and serum markers of HBV antigens and anti-HBV antibodies are indicated on the top panel of each figure. LDL, lower detectable limit. Gastroenterology 2008 134, 1938-1949.e3DOI: (10.1053/j.gastro.2008.03.037) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 PD-1 up-regulation favors HBV-specific CD8 T-cell apoptosis in patients with AHB. (A) Representative histograms indicate that PD-1–positive cells have higher levels of annexin V staining than PD-1–negative cells in the early phase of patients with AHB. (B) Pooled data indicate the annexin V–positive cell percentages in HBV-specific PD-1–positive and PD-1–negative CD8 T-cell subsets. (C) Representative PD-L1 or PD-L2 expression on CFSE-labeled HepG2215 cells at different time points (n = 3). Histogram values represent the PD-L1 or PD-L2 percentage on HepG2215 cells. (D) Representative dot plots for apoptosis of CD8 T cells from patients with AHB induced by HepG2215 cells with either isotype control or anti–PD-L1. Values in the upper right quadrant indicate the annexin V–positive CD8 T-cell percentages. (E) Pooled data show that blocking PD-1 ligation reduced annexin V–positive CD8 T-cell percentages in patients with AHB (n = 3). *P < .05 compared with blockade of PD-1 ligation. Gastroenterology 2008 134, 1938-1949.e3DOI: (10.1053/j.gastro.2008.03.037) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 PD-1 up-regulation restrains HBV-specific CD8 T-cell proliferation in patients with AHB. (A) Representative dot plots of pentamer percentage (upper right quadrant) from a patient with AHB at clinical presentation. (B) Summary data for the fold increase of pentamer percentage with peptide and either isotype control or anti–PD-L1. (C) Representative dot plots of a CFSE experiment using anti–PD-L1 are shown for a patient with AHB at clinical presentation. Values in the upper left quadrant indicate the percentage of CFSElow pentamer cells. Data are representative of 4 independent individuals. Gastroenterology 2008 134, 1938-1949.e3DOI: (10.1053/j.gastro.2008.03.037) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 PD-1 up-regulation regulates the cytokine production of HBV-specific CD8 T cells in patients with AHB. (A) Representative percentage of pentamer cells expressing intracellular IFN-γ (upper right quadrant) from a patient with AHB at clinical presentation. (B) Summary data for the percentage of IFN-γ–producing pentamer cells with peptide and either isotype control or anti–PD-L1. (C and D) Cytometric bead assay was performed on 10-day supernatants of the proliferation assay to quantify cytokine production with peptide and anti–PD-L1 or isotype control at clinical onset of acute HBV infection. Horizontal bars indicate the median values. Gastroenterology 2008 134, 1938-1949.e3DOI: (10.1053/j.gastro.2008.03.037) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Schematic patterns of dynamic PD-1 expression in relation to the outcome of acute HBV infection. Following HBV infection, HBV rapidly replicates and peaks within 4–6 weeks of infection, while most of the HBV DNA can be cleared from the serum before the ALT peaks after approximately 10–15 weeks after infection.1,40 Paralleling the serum ALT peak, PD-1 expression also rises to a peak during the acute symptom phase. This early transitory PD-1 up-regulation may efficiently temper overaggressive liver injury through restraining excessive T-cell responses and correlates with acute self-limited hepatitis B (A). In contrast, some primary infection develop into viral persistence, although the early events unfold as in self-limited infection.1,40 PD-1 might also be persistently highly expressed on effector CD8 T cells leading to T-cell exhaustion,11 thus correlating with chronic hepatitis B (B). Shadows indicate the examined time phases of this study. Dashed lines indicate conjectural alteration. Gastroenterology 2008 134, 1938-1949.e3DOI: (10.1053/j.gastro.2008.03.037) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure PD-1 expression does not associated with plasma HBV DNA level in the early phase of acute HBV infection. (A) There are no correlations between the PD-1 expression (percentage, left; MFI, right) on total CD8 T cells (A) or HBV-specific CD8 T cells (B) and plasma HBV DNA level in early phase of 23 AHB patients. (C and D) There are no correlations between the percentage of pentamer cells and plasma HBV DNA (C) or serum ALT levels (D) in early phase of acute HBV infection. solid line, linear growth trend; r, correlative coefficient. P values are shown Gastroenterology 2008 134, 1938-1949.e3DOI: (10.1053/j.gastro.2008.03.037) Copyright © 2008 AGA Institute Terms and Conditions