Reproducibility of T cell responses to HbsAg. PBMCs from 4 donors were independently purified by density gradient centrifugation and frozen by two different operators. PBMCs were then thawed and cultured with a HBsAg peptide pool or vehicle for 18 hours, in the presence of brefeldin A and the expression of IL-2, INF-γ and TNF-α in CD3+CD4+ and CD3+CD8+ cells was determined by specific staining and flow cytometry analysis. Background cytokine expression from unstimulated cells was subtracted from HBsAg peptide-stimulated cells. Percentages of CD4+ (panel A) and CD8+ (panel B) T cells 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+ obtained for the four donors in two independent experiments are presented.