1. Volume 143, Issue 6, Pages 1576-1585.e4 (December 2012)
Combined Blockade of Programmed Death-1 and Activation of CD137 Increase Responses of Human Liver T Cells Against HBV, But Not HCV 
Paola Fisicaro, Caterina Valdatta, Marco Massari, Elisabetta Loggi, Lara Ravanetti, Simona Urbani, Tiziana Giuberti, Albertina Cavalli, Carmen Vandelli, Pietro Andreone, Gabriele Missale, Carlo Ferrari 
Gastroenterology 
Volume 143, Issue 6, Pages e4 (December 2012)
DOI: /j.gastro
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2. Figure 1
Cytokine production by HBV- and HCV-specific T cells after in vitro expansion. Lymphocytes isolated from liver and peripheral blood of chronic HBV and HCV patients were expanded by stimulation with 15-mer peptide pools, spanning the HBV core region and the HCV-NS3 protein. Cytokine production was tested by intracellular cytokine staining. (A) Percentage of CD8+ and CD4+ producing IFN-γ or IL-2 for each patient (statistical comparisons by the Wilcoxon-paired test and the Mann–Whitney U test). (B) Inverse correlation by the Spearman rank correlation test between the percentage of IFN-γ+ HBV-specific T cells (CD8+CD4) and serum HBV DNA.
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3. Figure 2
Effect of PD-1 and CD137 manipulations on cytokine production by intrahepatic T cells. LIL from HBV (10 HBeAg− and 3 HBeAg+) and HCV (17) patients were cultured in the presence of an anti–PD-L1 antibody and/or a recombinant CD137L, or in the absence of both, as control. The frequency of cytokine-producing CD8+ and CD4+ cells was determined by intracellular cytokine staining. For each patient the ratio between the amount of cytokine produced in the different experimental conditions and in the control cultures (fold increase) was calculated. A fold increase greater than 1.5 defined responding patients. (A) Plots show fold increase values for each patient. Above each plot, the numbers and percentages of responsive patients are reported. (B) Comparison between results in HBV and HCV patients, expressed as percentages of responsive patients (Fisher exact test) and also as mean fold increases (Mann–Whitney U test), considering the total cytokine production by the overall T-cell population (CD4+CD8 cells).
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4. Figure 3
Effect of CD137L and the association of anti–PD-L1+CD137L with respect to anti–PD-L1 alone. (A–C) Histograms show the ratio between the mean fold increase of cytokine production by T cells of the overall HBV- or HCV-infected populations observed in the presence of CD137L, or anti–PD-L1+CD137L, with respect to the mean fold increase in the presence of anti–PD-L1 alone. On the bottom, representative dot plots displaying IFN-γ, IL-2, and IFN-γ/IL-2 production. (D) Left plot: percentages of patients responding with a concomitant increment of INF-γ+, IL-2+, and IFN-γ+IL-2+ cells to the different manipulations. Right plot: percentages of patients not responding at all.
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5. Figure 4
Effect of PD-1 and CD137 manipulations on the peripheral T-cell function. Comparison between peripheral HBV- and HCV-specific T cells (CD4+CD8) relative to their production of INF-γ, IL-2, and IFN-γ/IL-2, expressed as the percentage of responsive patients (Fisher exact test) and as the mean fold increase (Mann–Whitney U test). Significant differences (Wilcoxon-paired test) between fold increase values observed in anti–PD-L1 and anti–PD-L1+CD137L–treated HCV cultures also are shown.
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6. Figure 5
Expression of co-stimulatory molecules on virus-specific CD8+ T cells. Virus-specific CD8+ cells were detected through co-staining with pools of class I tetramers and antibodies to the indicated co-stimulatory molecules. (A) Frequencies of intrahepatic HBV- and HCV-specific CD8 cells; (B) expression of known costimulatory molecules on intrahepatic tetramer+ CD8 cells (Mann–Whitney U test). (C) Representative dot plots showing CD137 expression on HBV and HCV tetramer+ CD8 T cells. (D) Mean values of expression of the different co-stimulatory molecules on intrahepatic and peripheral virus-specific CD8 cells from HBV- and HCV-infected patients (Wilcoxon paired test).
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7. Figure 6
Frequency and phenotype of regulatory CD4+FoxP3+ T cells. Top panel: frequencies of intrahepatic and peripheral CD4+FoxP3+ T cells. Bottom panel: phenotype of Tregs derived from HBV- and HCV-infected livers. Statistical comparisons by Mann–Whitney U test and Wilcoxon paired test.
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8. Supplementary Figure 1
CD137L dose-titration experiments with CD137L. Representative experiments performed on PBMCs from chronic HBV and HCV patients are shown with the aim of identifying the optimal CD137L dose able to give the best level of T-cell stimulation. (A) Tetramer expansion at graded CD137L concentrations. (B) Tetramer expansion and the corresponding percentage of annexin V+/7-aminoactinomycin D (7-AAD)+ cells at the indicated CD137 doses.
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9. Supplementary Figure 2
Effect of PD-1 and CD137 manipulations on in vitro expansion of intrahepatic and peripheral virus-specific CD8+ T cells analyzed by tetramer staining. Intrahepatic and peripheral blood lymphocytes from HLA-A2+ HBV and HCV patients were cultured in vitro in the presence of an anti–PD-L1 antibody and/or of a recombinant CD137L, and in the absence of both as control. Pools of HLA-A2–restricted HBV or HCV peptides were used for culture stimulation, and the corresponding HLA class I tetramers were pooled for staining. The upper part of panel A illustrates 2 HLA-A2+ HBV liver samples with percentages of HBV-specific CD8 cells induced by each experimental condition within circles. The only case of tetramer+ cell expansion among liver HCV samples is reported on the bottom. Panel B illustrates the in vitro expansion of peripheral lymphocytes from 2 representative patients, one infected by HBV and the other by HCV. ND, not determined.
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10. Supplementary Figure 3
Effect of different CD137L doses on anti–PD-L1–mediated functional T-cell restoration. (A and B) Two examples of HBV or HCV tetramer+ cell expansion with the corresponding frequency of annexin V+/7-aminoactinomycin D+ cells. (C and D) Two examples of cytokine production by T-cell stimulation with (C) HBV or (D) HCV peptide pools. As in panels A and B, an improvement of the anti–PD-L1 effect is observed at CD137 concentrations lower than 1 μg/mL.
Gastroenterology  , e4DOI: ( /j.gastro )
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11. Supplementary Figure 4
Effect of PD-1 and CD137 manipulations on cytokine production by peripheral HBV- and HCV-specific T cells. Peripheral T cells from HBV or HCV patients were cultured in vitro in the presence of an anti–PD-L1 antibody and/or recombinant CD137L, and in the absence of both as control. The ratio between the amount of cytokine produced in the different conditions and in the control culture (fold increase) was calculated for each patient. A fold increase greater than 1.5 was used to define responding patients. Each circle corresponds to a single patient. Above the plots, numbers and percentages of responsive patients are reported.
Gastroenterology  , e4DOI: ( /j.gastro )
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