1. Volume 133, Issue 4, Pages 1132-1143 (October 2007)
HCV-Specific T-Cell Response in Relation to Viral Kinetics and Treatment Outcome (DITTO-HCV Project) 
Massimo Pilli, Alessandro Zerbini, Amalia Penna, Alessandra Orlandini, Esther Lukasiewicz, Jean–Michel Pawlotsky, Stefan Zeuzem, Solko W. Schalm, Michael von Wagner, Georgios Germanidis, Yoav Lurie, Juan I. Esteban, Bart L. Haagmans, Christophe Hezode, Martin Lagging, Francesco Negro, Yonit Homburger, Avidan U. Neumann, Carlo Ferrari, Gabriele Missale 
Gastroenterology 
Volume 133, Issue 4, Pages (October 2007)
DOI: /j.gastro
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2. Figure 1
Correlation between HCV-specific proliferative or CD8-mediated T-cell responses and slope of viral decay during the first month of treatment (slope of HCV-RNA decay days 1–29). For proliferative responses (A), SI values obtained with recombinant proteins at weeks 2 and 4 after start of therapy were summed and the resulting value was subtracted from the value obtained by summing SI values of weeks −2 and −4 before treatment. Each value then was plotted against the corresponding value of viral decay. Viral decay was calculated by the slope formula expressing the steepness of the line defined by the viral load measurements between days 1 and 29 of treatment. Similarly, (B) HCV-specific CD8 cell frequencies analyzed ex vivo or after in vitro expansion (C) and frequencies of HCV-specific IFN-γ–positive CD8 cells obtained in each patient during the first month of treatment subtracted from the CD8 cell frequencies and IFN-γ responses measured before treatment (D) also were plotted against viral decay. Therefore, each dot represents the modification of the HCV-specific T-cell response in relation to the decay of viral load during the first month of treatment. None of the linear regressions were statistically significant (Spearman correlation coefficient). HLA class-I tetramers as well as HLA-A2 and HLA-A3–restricted peptides were used for CD8 analysis.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
HCV-specific proliferative and CD8-mediated T-cell responses before and during the first month of treatment in patients with different viral kinetics. (A) Frequency of significant (SI ≥ 3) proliferative T-cell responses (tested in 22 patients with 4 different recombinant HCV proteins) calculated on all tests performed at each time point, (B) frequency of HCV-specific CD8 cells ex vivo, and (C) after 10 days of in vitro expansion (tested in 27 patients), (D) frequency of IFN-γ–positive CD8 cells on restimulation of short-term T-cell lines (tested in 27 patients), and (E) mean frequency of positive cytotoxic T-cell responses (showing at least 10% of specific target lysis, tested in 22 patients) are illustrated. For CTL analysis, 104 CTL lines (62 derived from RVR and 42 from non-RVR patients) were tested. Frequencies of positive cytotoxic tests in RVR and non-RVR patients were not significantly different (χ2 analysis). □ and ○, T-cell responses in RVR patients,■ and ●, T-cell responses in non-RVR patients. Frequencies of tetramer-positive cells measured ex vivo at week −1 (P = .01) and week 2 (P = .01) and frequencies of tetramer-positive cells measured after in vitro expansion at week 2 (P < .05) were significantly different in RVR and non-RVR patients. Recombinant HCV proteins were used to analyze proliferative T-cell responses, whereas HLA class-I tetramers and HLA-A2 and HLA-A3–restricted peptides were used for CD8 analysis. Tetramer and IFN-γ staining values below the cut-off level were considered equal to 0 for statistical analysis (Mann–Whitney).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
Tetramer staining analysis of CD8 responses. PBMCs derived from 4 representative patients were stained with HLA class I tetramers ex vivo and after 10 days in vitro expansion by stimulation with HLA-A2 and HLA-A3 9-mer peptides 4 and 2 weeks before and 2 and 4 weeks after the start of treatment. CD8 cells from patient 6006 were stained with tetramer HLA-A2–NS , CD8 cells from patient 7022 and 6035 with tetramer HLA-A3–NS , and CD8 cells from patient 6023 with tetramer HLA-A3–NS
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
IFN-γ ELISPOT analysis of HCV-specific T-cell responses. PBMCs from 4 RVR patients were stimulated for 10 days with 25 pools of 15-mer peptides representing the entire HCV-1 sequence. Derivative short-term T-cell lines then were washed extensively and seeded in an ELISPOT plate either with medium (control) or with the appropriate peptide pools for 18 hours. Numbers of spots obtained with medium (mean value, 93 ± 97 spots/2 × 105 cells) were subtracted from the spots generated in the peptide restimulated wells (delta). Bars represent the average delta spots of the different T-cell lines measured at the indicated time points, the broken line with black square symbols shows the frequency of positive T-cell responses (at least 1.5-folds the number of spots generated with medium alone) at each time point.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
HCV-specific CD8 cell response measured before and during the first month of treatment in genotype-1–infected patients with different treatment outcomes. Frequencies of HCV-specific CD8 cells measured (A) ex vivo or on (B) short-term T-cell lines in 12 SVR (Δ) and 10 non-SVR patients (▲) are shown. (C) Frequencies of IFN-γ–positive CD8 cells in short-term T-cell lines derived from 11 SVR (Δ) and 9 non-SVR patients (▲). Frequencies of tetramer-positive cells measured after in vitro expansion at week −4 (P < .005), week −1 (P < .005), week 2 (P = .001), and week 4 (P = .05) were significantly different in SVR and non-SVR patients (Mann–Whitney U test). (D) The basal (weeks −4 and −1) frequency of tetramer-positive CD8 cells after in vitro expansion was plotted against HCV viral load measured at the same time points. Recombinant HCV proteins were used to analyze proliferative T-cell responses, whereas HLA class-I tetramers and HLA-A2 and HLA-A3–restricted peptides were used for CD8 analysis. Tetramer and IFN-γ staining values below the cut-off level were considered equal to 0 for statistical analysis (Mann–Whitney).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
HCV-specific proliferative and CD8-mediated T-cell responses before treatment, during the whole treatment period, and during the follow-up period in patients randomized to the standard treatment. (A) Frequency of significant (SI > 3) proliferative T-cell responses analyzed in 13 patients. Average frequencies of tetramer-positive CD8 cells measured (B) ex vivo and (C) after in vitro expansion in 12 patients. HCV-specific CD8 cells were not detected by tetramer staining, both ex vivo and after short-term in vitro expansion, in only 2 patients. (D) Frequency of significant (target lysis > 10%) cytotoxic T-cell responses (analyzed on 38 in vitro expanded T-cell lines) and (E) average frequencies of IFN-γ–positive CD8 cells on restimulation of short-term T-cell lines are shown. (F) The effect of mitogen stimulation on IFN-γ production by CD8 cells, performed in 11 patients, is shown. Recombinant HCV proteins were used to analyze proliferative T-cell responses, whereas HLA class-I tetramers and HLA-A2 and HLA-A3 restricted peptides were used for CD8 analysis.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions