Expression of activation markers on the T-cell surface. The expression of several molecules involved in T-cell activation was analyzed by flow cytometry during the three rounds of stimulation (S1 to S3). T cells were cultured with MV (shaded bars)- or LV (filled bars)-infected iDC or mDC for the first stimulation and were restimulated twice with inactivated-MV- or inactivated-LV-pulsed DC, respectively. The percentage of T cells cultured with infected DC and restimulated with inactivated-virus-pulsed DC that express the markers minus the percentage of T cells cultured and restimulated with mock-infected DC and restimulated with culture medium that express the markers was calculated. Results are means and SEM from five independent experiments using different donors. Significant differences between mock-infected and infected cells are indicated as for Fig. 1. The expression of the respective marker in control T cells that had first been stimulated with mock-infected iDC was quantified 2 and 5 days after restimulation with DC pulsed with inactivated MV (open circles on shaded bars) or with inactivated LV (open circles on filled bars) and is expressed as the mean from two independent experiments using different donors. (A) The expression of CD154 on CD3+ CD4+ (top) and CD3+ CD8+ (bottom) T cells cultured with iDC or mDC is shown for 5 days after each stimulation. (B) CD154 mRNA synthesis was evaluated by qRT-PCR 2 days after each round of stimulation in T cells cultured with mock (open bars)-, MV (shaded bars)-, or LV (filled bars)-infected iDC. The means and SEM from five independent experiments using different donors are shown. Results are expressed as the gene/β-actin ratio. (C and D) The expression of CD137 (C) and HLA-DR (D) by CD3+ CD8+ and CD3+ CD4+ T cells cultured with iDC or mDC is shown for day 2 (d2) and d5 of the three rounds of stimulation. Dot plots showing the expression of CD8 and CD137 or of CD4 and CD154 or HLA-DR among CD3-gated cells 5 days after the second stimulation with mock-, MV-, or LV-infected iDC are presented.