Percentage of CD4+ T cells expressing IFN-γ, TNF-α and/or IL-2 upon HBsAg-peptide stimulation. PBMC were isolated from whole blood by density gradient and frozen. Following completion of the protocol, cells were thawed and cultured with 22 HBsAg 20-mers peptides or with vehicle for 18 hours, in the presence of brefeldin A. PBMC were then fixed, permeabilized and stained with fluorochrome-conjugated CD3, CD4, CD8, IFN-γ, TNF-α, IL-2 specific antibodies. The percentage of CD3+CD4+ T lymphocytes expressing IFN-γ, TNF-α and/or IL-2 was determined by flow cytometry. Background cytokine expression from unstimulated cells was subtracted from HBsAg peptides-stimulated cells. Percentages of CD4+ lymphocytes either IL-2- IFN-γ+ TNF-α-, IL-2- IFN-γ- TNF-α+, IL-2+ IFN-γ- TNF-α-, IL-2- IFN-γ+ TNF-α+, IL-2+ IFN-γ- TNF-α+, IL-2+ IFN-γ+ TNF-α- or IL-2+ IFN-γ+ TNF-α+ are presented in groups Engerix-B® (panel A), HBsAg alone (panel B), HBsAg + 3 μg IMP321 (panel C), HBsAg + 10 μg IMP321 (panel D), HBsAg + 30 μg IMP321 (panel E) and HBsAg + 100 μg IMP321 (panel F) for every subject at every time point (see x-axis). Statistically significant increases (p < 0.05) are shown by an asterisk.