1. Volume 138, Issue 2, Pages 682-693.e4 (February 2010)
Antiviral Intrahepatic T-Cell Responses Can Be Restored by Blocking Programmed Death-1 Pathway in Chronic Hepatitis B 
Paola Fisicaro, Caterina Valdatta, Marco Massari, Elisabetta Loggi, Elisabetta Biasini, Luca Sacchelli, Maria Cristina Cavallo, Enrico M. Silini, Pietro Andreone, Gabriele Missale, Carlo Ferrari 
Gastroenterology 
Volume 138, Issue 2, Pages e4 (February 2010)
DOI: /j.gastro
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2. Figure 1
Frequency of HBV-specific CD8+ T cells in liver and peripheral blood of chronic HBV patients. Liver infiltrating and peripheral blood HBV-specific CD8+ cells from 17 HLA-A2+ chronic HBV patients were stained with a pool of 6 HLA-A2 tetramers. Panel A illustrates percentages of tetramer-positive cells on total CD8 T cells. Differences between intrahepatic and peripheral tetramer-positive cell frequencies were assessed by the Wilcoxon paired test. Panel B represents mean frequency values of intrahepatic tetramer-positive CD8 cells subdivided in relation to different ranges of HBV-DNA concentration. P values according to the Mann–Whitney test are reported. Panel C shows the inverse correlation between percentages of intrahepatic tetramer-positive CD8 cells and serum HBV-DNA titers, assessed by the Spearman test.
Gastroenterology  , e4DOI: ( /j.gastro )
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3. Figure 2
PD-1 and CD127 expression by tetramer-positive and total CD8 T cells. Panel A: Percentages of PD-1+ and CD127+ cells among HBV-tetramer+ CD8 lymphocytes isolated from liver or blood of chronic HBV patients. Dot plots on the right show a representative example of intrahepatic and peripheral blood HBV-specific CD8 cells. Panel B: Correlation between PD-1+ intrahepatic tetramer+ CD8 cells and Ishak hepatic fibrosis score (P = .01 by the Spearman test). Panel C: PD-1 (left) and CD127 (right) expression on HBV-specific and total CD8 cells derived from liver and blood. Panel D: Expression of PD-1 and CD127 on intrahepatic and peripheral CMV-specific CD8 cells; all CMV-responsive patients were positive for serum IgG but not for IgM anti-CMV antibodies. A representative example is illustrated in the right dot plots. Statistical analysis in panels A and C was carried out on a paired set of data (intrahepatic and peripheral frequencies of HBV-specific and total CD8 cells) by the Wilcoxon paired test.
Gastroenterology  , e4DOI: ( /j.gastro )
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4. Figure 3
PD-1 expression is higher on intrahepatic than on peripheral lymphocytes at all T-cell differentiation stages. Frequencies of intrahepatic and peripheral HBV-specific (panel A) and total (panel B) CD8 cell subsets with different CD45RA and CCR7 expression. Histograms illustrate the percentage of PD-1+ T cells in each T-cell subset. Differences between intrahepatic and peripheral PD-1 expression were assessed by the Wilcoxon paired test. Reported values derive from 12 patients.
Gastroenterology  , e4DOI: ( /j.gastro )
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5. Figure 4
Anti-PD-L1 can enhance the capacity of HBV-specific T cells to produce IFN-γ. Lymphocytes from liver and peripheral blood were cultured in vitro in the presence of anti-PD-L1 or an isotypic control antibody and IFN-γ production was analyzed by intracellular cytokine staining upon stimulation with a mixture of overlapping 15mer peptides covering the overall HBV-core or HBV-polymerase regions. Panel A illustrates the effect on cytokine production of liver and blood CD8 and CD4 cell induced by anti-PD-L1. Panel B depicts changes in frequency of IFN-γ producing CD8 and CD4 cells analyzed together induced by anti-PD-L1, assessed as the ratio between the percentage of IFN-γ-positive T cells detected in cultures treated with anti-PD-L1 and the percentage detected in control cultures (fold increase). Responsive patients with fold increase >1.5 are illustrated. Statistics were done by the Wilcoxon paired test.
Gastroenterology  , e4DOI: ( /j.gastro )
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6. Figure 5
Anti-PD-L1 can improve IL-2 production by HBV-specific T cells. Liver-derived and peripheral blood T cells were stimulated with HBV-core or HBV-polymerase peptides in the presence of an anti-PD-L1 or an isotypic control antibody and were tested by intracellular cytokine staining for IL-2 production. Panel A illustrates the changes in IL-2 production detected in anti-PD-L1 compared with control antibody treated cultures. Panel B shows the overall effect of anti-PD-L1 on CD8 and CD4 cells represented together, as assessed by fold increase in IL-2 producing cells. Responsive patients with fold increase >1.5 are illustrated.
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7. Figure 6
Effect of anti-PD-L1 on IFN-γ and IL-2 production by intrahepatic HBV-specific T cells analyzed by tetramer staining. Intrahepatic lymphocytes from 4 HLA-A2+ chronic HBV patients were cultured in the presence of anti-PD-L1 or a control antibody and stimulated with a pool of HLA-A2 restricted HBV peptides (core, 18–27; envelope 183–191, 335–343, and 348–357; polymerase, 575–583 and 816–824). The Figure illustrates in vitro expansion and cytokine production upon anti-PD-L1 or control antibody treatment by tetramer-positive CD8 cells.
Gastroenterology  , e4DOI: ( /j.gastro )
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8. Figure 7
Effect of anti-PD-L1 on IFN-γ and IL-2 production by intrahepatic CMV- and FLU-specific T cells analyzed by tetramer staining. Liver infiltrating lymphocytes from 4 HLA-A2+ patients were cultured in the presence of anti-PD-L1 or a control antibody and stimulated with HLA-A2 restricted CMV or FLU peptides. T-cell expansion and cytokine production are illustrated.
Gastroenterology  , e4DOI: ( /j.gastro )
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9. Figure 8
IFN-γ production by liver infiltrating T cells is more efficiently improved by anti-PD-L1 at low HBV-DNA titers. IFN-γ production by liver infiltrating CD8 and CD4 cells after anti-PD-L1 treatment was analyzed in relation to patients' HBV-DNA titers. Statistical analysis was done by the Wilcoxon paired test to compare the effect of the anti-PD-L1 and the control antibodies and by the Mann–Whitney test to assess the differences between cytokine recovery in patients with an HBV-DNA titer below or above 1 × 106copies/mL.
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10. Supplementary Figure 1
PD-1 and CD127 expression by intrahepatic HBV-specific CD8 T cells. Representative dot plots showing tetramer-positive cell frequency as well as PD-1 and CD127 expression on HBV-specific CD8 cells isolated from the liver of 8 chronic HBV patients.
Gastroenterology  , e4DOI: ( /j.gastro )
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11. Supplementary Figure 2
PD-1 mean fluorescence intensity (MFI) is higher on intrahepatic than peripheral CD8 cells. Representative examples of PD-1 MFI of HBV-specific (upper panel) and total (lower panel) CD8 cells from liver and blood are illustrated on the left of each panel. Histograms on the right of each panel illustrate mean values of PD-1 MFI from all patients. Statistical analysis was performed by the Wilcoxon paired test.
Gastroenterology  , e4DOI: ( /j.gastro )
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12. Supplementary Figure 3
PD-1 expression on intrahepatic T-cell subsets. Frequencies of PD-1-positive CD8 cells among T-cell subsets identified by different expression of CD45RA and CCR7. Panel A illustrates HBV-specific CD8 cells, whereas total CD8 cells are depicted in panel B. The highest PD-1 expression is displayed by CD45RA+CCR7+ and CD45RA−CCR7+ cells.
Gastroenterology  , e4DOI: ( /j.gastro )
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13. Supplementary Figure 4
Effect of anti-PD-L1 on IFN-γ and IL-2 production by peripheral blood CMV- and FLU-specific CD8 cells analyzed by tetramer staining. Circulating lymphocytes from 3 HLA-A2+ chronic HBV patients were cultured in the presence of anti-PD-L1 or a control antibody and stimulated with HLA-A2 restricted CMV or FLU peptides. Expansion and cytokine production are illustrated.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions