1. Volume 143, Issue 4, Pages 963-973.e9 (October 2012)
Restored Function of HBV-Specific T Cells After Long-term Effective Therapy With Nucleos(t)ide Analogues 
Carolina Boni, Diletta Laccabue, Pietro Lampertico, Tiziana Giuberti, Mauro Viganò, Simona Schivazappa, Arianna Alfieri, Marco Pesci, Giovanni B. Gaeta, Giuseppina Brancaccio, Massimo Colombo, Gabriele Missale, Carlo Ferrari 
Gastroenterology 
Volume 143, Issue 4, Pages e9 (October 2012)
DOI: /j.gastro
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2. Figure 1
IFN-γ, IL-2, and TNF-α production by HBV-specific CD4 and CD8 T cells after in vitro expansion. Cytokine production was analyzed by ICS after 10 days of stimulation with different HBV peptide pools. (A) Data are represented as the mean frequency of CD4 and CD8 T cells producing IFN-γ, IL-2, or TNF-α (showed by black, gray, and white bars, respectively) to peptide pools spanning distinct HBV regions. In B, black circles and triangles represent the total frequency of IFN-γ–positive (upper graph) and IL-2–positive (lower graph) HBV-specific CD4 and CD8 cells detected in each tested patient and obtained by summing responses to individual peptide pools. (C) Mean of the global frequencies of cytokine-producing CD4 and CD8 cells for each patient population indicated with the same symbols used in A. Differences in global CD4- and CD8-mediated responses within the different patient groups by the Wilcoxon matched pairs test were as follows: P = .03 in naïve HBeAg-negative CHB, P = .04 in inactive carriers, P = in spontaneous resolvers of chronic infection, P = .006 in NUC-treated HBsAg-positive patients, P = .009 in NUC-treated HBsAg-negative patients.
Gastroenterology  , e9DOI: ( /j.gastro )
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3. Figure 2
Polyfunctionality of HBV-specific T cells. Cytokine production by HBV-specific CD3+ T cells was analyzed after 10 days of in vitro stimulation with HBV peptide pools. (A) Data are represented as mean frequency of HBV-specific CD3+ T cells able to produce simultaneously the indicated cytokines in response to all peptide pools (plus SE) in different patient categories. (B) Representative dot plots from 4 NUC-treated patients. Statistically significant differences between levels of cytokine production in different patient populations, as determined by the Mann–Whitney test, are indicated.
Gastroenterology  , e9DOI: ( /j.gastro )
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4. Figure 3
Phenotypic profiles of virus-specific CD8+ T cells. (A) Expression of CD45RA and CCR7 surface molecules was assessed directly ex vivo on HBV-, Flu-, and CMV-specific CD8 cells in different patient categories by using HLA class I dextramers. Each black circle, white circle, and white triangle represents the frequency of CD45RA−/CCR7− (upper graph) and CD45RA+/CCR7− (lower graph) HBV-, Flu-, and CMV-specific CD8 T cells, respectively, in each tested patient. HBV-specific T cells derive from patients 7a, 12a, 14a, 19b, 22–25b, 1d, 12–14d, 5f, and 9–12f (Tables 1 and 2 and Supplementary Tables 2 and 4); patients 7a and 14d were positive for 2 distinct epitopes, and patient 12d was tested at 2 distinct time points. Flu- and CMV-specific T cells derive from NUC-treated patients. Statistically significant differences between phenotype profiles in different patient populations, as determined by the Mann–Whitney test, are indicated. (B) Representative dot plots from different patients.
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5. Figure 4
(A) Ex vivo functional profile of specific CD8+ T cells. IFN-γ, IL-2, and TNF-α production and degranulation potential (CD107a), assessed ex vivo on HBV, Flu, and CMV dextramer-positive cells following peptide stimulation, are shown for each individual among different patient categories. HBV-specific T cells were tested in patients 7a, 12a, 14a, 19b, 22b, 23b, 25b, 1d, 12–14d, 5f, and 9–12f (Tables 1 and 2 and Supplementary Tables 2 and 4); patients 1d and 5f were tested at 2 distinct time points, and patient 13d was tested at 3 distinct time points. Flu- and CMV- specific T cells derive from NUC-treated patients. (B) Dot plots from representative patients. (C) Mean values (plus SE) of IFN-γ–secreting cells after 18 hours of stimulation with the overall HBV peptide panel, assessed by ELISPOT assay. (Inset) The percentage of positive responses on total tests for the same patient categories. ELISPOT was considered positive if the number of spots in the stimulated wells was at least 2 times greater than background and the difference between the number of spots in the stimulated and unstimulated wells was >10. The mean spot-forming cells in the unstimulated wells was generally <10. Statistically significant differences detected by the Mann–Whitney test are indicated.
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6. Figure 5
Functional profile of HBV-specific CD8+ T cells after in vitro expansion. IFN-γ and IL-2 production, (A) degranulation potential (CD107a), and (B) expansion capacity expressed by individual patients belonging to distinct patient categories were analyzed by ICS on dextramer-positive CD8 cells after 10 days of in vitro culture. Fold increase represents the ratio between frequencies ex vivo and after 10 days of peptide stimulation of HBV-specific CD8 cells detected by dextramer staining. (C) Dot plots from 3 representative NUC-treated patients.
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7. Supplementary Figure 1
T-cell responses to HBV peptides after in vitro expansion in 2 representative patients. Dot plots of ICS experiments with the overall peptide panel composed of 16 peptide pools in 2 representative NUC-treated patients. The X region was covered by peptide pools 1 and 2, the core region by 3 and 4, the envelope region by 5–8, and the polymerase region by 9–16.
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8. Supplementary Figure 1
T-cell responses to HBV peptides after in vitro expansion in 2 representative patients. Dot plots of ICS experiments with the overall peptide panel composed of 16 peptide pools in 2 representative NUC-treated patients. The X region was covered by peptide pools 1 and 2, the core region by 3 and 4, the envelope region by 5–8, and the polymerase region by 9–16.
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9. Supplementary Figure 2
IFN-γ, IL-2, and TNF-α production by HBV unrelated antigen-specific CD4 and CD8 T cells. Cytokine production was analyzed in 4 NUC-treated patients by ICS after 10 days of stimulation with irrelevant HCV peptides (3 pools of 15 mer peptides spanning HCV sequences 1–310, 2401–2710, and 2701–3011) and with a panel of 9–21 mer HLA class I– or class II–restricted peptides containing relevant CMV, Epstein–Barr virus, Flu, and tetanus toxoid epitopes. Data are represented as the mean frequencies of individual cytokine CD4 and CD8 T-cell responses (plus SE) stimulated by distinct peptide pools.
Gastroenterology  , e9DOI: ( /j.gastro )
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10. Supplementary Figure 3
Patterns of CD4 and CD8 functions expressed by different patient categories. Following calculation of the total frequency of IFN-γ–, IL-2–, and TNF-α–positive cells among the overall CD4 and CD8 populations, as determined by summing the response to individual peptide pools in each individual patient, 3 threshold levels were defined by calculating the 33rd, 66th, and 99th percentiles. Three different functional patterns were identified according to the percentile levels of IFN-γ, IL-2, and TNF-α production: pattern 1 (values above the 66th percentile) identifies patients with broader and higher cytokine production (++/++/++, ++/++/+, +/++/++), pattern 2 (values between the 33rd and the 66th percentiles) with cytokine production of intermediate breadth and strength (+/+/+, ++/+/−, +/−/++, +/+/−, −/+/+, +/−/+, +/+/++), and pattern 3 (values below the 33rd percentile) with narrower and lower cytokine production (−/−/−,−/−/+,−/+/−, +/−/−). Each bar illustrates the percentage of patients expressing individual functional patterns for CD8 and CD4 T-cell responses.
Gastroenterology  , e9DOI: ( /j.gastro )
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11. Supplementary Figure 4
Expression of PD-1 and CD127 by specific CD8+ T cells. Expression of PD-1 and CD127 surface molecules was assessed ex vivo on HBV-, Flu-, and CMV-specific CD8 cells from different patient categories identified by dextramer staining. Each black circle, white circle, and white triangle represents the frequency of PD-1+ (upper graph) and CD127+ (lower graph) HBV-, Flu- and CMV-specific T cells, respectively, in each tested patient. HBV-specific T cells were derived from patients 7a, 12a, 14a, 19b, 22–25b, 1d, 12–14d, 5f, and 9–12f (Tables 1 and 2); patients 7a and 14d were positive for 2 distinct epitopes, and patient 12d was tested at 2 distinct time points. Flu- and CMV-specific T cells were derived from NUC-treated patients. Statistically significant differences between phenotype profiles in different patient populations, as determined by Man–Whitney test, are indicated.
Gastroenterology  , e9DOI: ( /j.gastro )
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