Volume 39, Issue 4, Pages 595-605 (October 2003) Transient restoration of anti-viral T cell responses induced by lamivudine therapy in chronic hepatitis B  Carolina Boni, Amalia Penna, Antonio Bertoletti, Vincenzo Lamonaca, Irene Rapti, Gabriele Missale, Massimo Pilli, Simona Urbani, Albertina Cavalli, Simona Cerioni, Ruggero Panebianco, Julian Jenkins, Carlo Ferrari  Journal of Hepatology  Volume 39, Issue 4, Pages 595-605 (October 2003) DOI: 10.1016/S0168-8278(03)00292-7

Fig. 1 Behavior of proliferative T cell responses to HBcAg and HBeAg throughout the study period. Each bar represents the median stimulation index of proliferative T cell responses to HBcAg (panel A) and HBeAg (panel B) detected at each of the indicated time points in the three groups of patients with different virologic response and in the whole population of 12 HBeAg± patients with CH-B treated with lamivudine. Depletion experiments showed that proliferation to HBV proteins and peptides was sustained by CD4 but not by CD8 cells (data not shown), confirming that the response studied was CD4-mediated. The stimulation index (SI) represents the ratio between the mean cpm obtained in the presence and absence of antigen. SI values above 4 were regarded as positive (>2 standard deviations above the mean SI value obtained with each individual HBV protein or peptide in normal controls). Data for each time point during therapy were compared with data of week 24 (preceding start of therapy) by the Student's t-test for paired data (*P=0.01–0.05; **P<0.01) in the whole group of patients where the sample size was sufficient to allow statistical comparison. Numbers above individual bars in the bottom panels represent the mean serum HBeAg concentration (ng/ml) detected in the overall patient population at the corresponding time points. Journal of Hepatology 2003 39, 595-605DOI: (10.1016/S0168-8278(03)00292-7)

Fig. 2 Longitudinal analysis of the T cell proliferative response to HBV core peptides in individual patients. The figure illustrates the response at sequential time points to only those peptides to which individual patients gave at least one significant response. At some time points responses were not tested because available PBMC were not sufficient to allow to perform all immunological assays simultaneously. Journal of Hepatology 2003 39, 595-605DOI: (10.1016/S0168-8278(03)00292-7)

Fig. 3 Proliferative T cell responses to HBV peptides. Each bar illustrates the percentage of significant proliferative T cell responses to the entire panel of peptides displayed by high (panel A), intermediate (panel B) and low (panel C) responders and by the whole patient population (panel D) at each of the indicated time points. Data for each time point during therapy were compared with data of week 24 (preceding start of therapy) by the Chi-Square test (*P=0.01–0.05; **P<0.01). The internal square in panel D represents the mean percentage of significant proliferative T cell responses to the whole panels of synthetic peptides expressed by the whole patient population before, during the first (1–6 months) and second half (7–12 months) of the treatment period and after therapy. Data before, during and after treatment were compared by the Chi-Square test. Journal of Hepatology 2003 39, 595-605DOI: (10.1016/S0168-8278(03)00292-7)

Fig. 4 Longitudinal analysis of the proliferative T cell response to HBV peptides before, during, after lamivudine therapy and during re-treatment in patient no. 8, who showed a reactivation of disease during the post-treatment follow-up with a sharp elevation of ALT (2500 U/ml) and bilirubin values, and a severe decline of protrombin activity. Journal of Hepatology 2003 39, 595-605DOI: (10.1016/S0168-8278(03)00292-7)

Fig. 5 IFN-γ production by T cells upon stimulation with HBcAg. Panel A. Dot-plot analysis of IFN-γ production by T cells derived from the indicated time points of a representative patient (patient no. 9) cultured in the presence or absence of HBcAg and stained with anti-IFN-γ and anti-IL4 monoclonal antibodies. Panel B. Number of IFN-γ positive lymphocytes among 105 total T lymphocytes in the whole patient population before, during and after lamivudine treatment. Since frozen cells were used for this study and not all time points were available for the whole patient population, some time points were grouped in order to have frequency data for each patient at all indicated time periods. Therefore, each bar represents the median T cell frequency of the whole patient group in the corresponding time periods. Data for each period during therapy were compared with data of the pre-treatment period by the Mann–Whitney U-test. A statistically significant difference was observed only for the 26–36 weeks period. Journal of Hepatology 2003 39, 595-605DOI: (10.1016/S0168-8278(03)00292-7)

Fig. 6 CTL responses to individual HBV peptides containing the HLA-A2 binding motif. Panel A. Each bar represents the percentage of significant cytotoxic responses to individual peptides (specific lysis >15%) among the total number of assays performed at different time points before, during the first and second half of treatment and after the end of therapy in the whole group of patients. Each patient was tested with each individual peptide four or five times before therapy, 11 or 12 times during lamivudine treatment and four times after therapy. Responses were HLA-A2 restricted because only HLA-A2+ but not HLA-A2− target cells pulsed with the relevant peptides were lysed by effector T cells (data not shown). Panel B. Percentage of significant CTL responses to the entire panel of HBV peptides expressed by each patient before, during the first and second half of treatment and after therapy. Statistical comparison was made by Chi-square test. Panel C. Each bar represents the percentage of significant CTL responses to the entire panel of peptides expressed by the whole patient population at each time point. Data for each time point during therapy were compared with data of week 24 (preceding start of therapy) by the Chi-Square test (*P=0.01–0.05; **P<0.01). The internal square in panel C represents the mean percentage of CTL responses expressed by the whole patient population before, during the first and second half of treatment and after therapy. Statistical comparison was made by Chi-square test. Journal of Hepatology 2003 39, 595-605DOI: (10.1016/S0168-8278(03)00292-7)

Fig. 7 Proliferative T cell response to PHA, tetanus toxoid and HBeAg displayed by 12 CH-B patients before, during and after lamivudine treatment. Each bar represents the mean stimulation index (Panel A) and the mean value of delta cpm (Panel B) of all patients at each time point. PBMC were cultured for 3 and 7 days to study proliferation to PHA and antigens, respectively. The stimulation index is the ratio between the mean cpm obtained in the presence and absence of antigen or mitogen; the delta cpm represents the cpm value derived from subtraction of the cpm of unstimulated from that of stimulated cultures. In contrast to mitogen and tetanus toxoid stimulated responses, the maximal level of cpm incorporation stimulated by HBV antigens increased during the initial time of therapy so that HBV-specific responses showed identical early elevation and later decline by both stimulation index and delta cpm evaluation. Journal of Hepatology 2003 39, 595-605DOI: (10.1016/S0168-8278(03)00292-7)

Journal of Hepatology 2003 39, 595-605DOI: (10 Journal of Hepatology 2003 39, 595-605DOI: (10.1016/S0168-8278(03)00292-7)