Fig. 7.

Induction of CTL by MV-infected DC. The expression of GrzB and perforin (perfo) at the surfaces of CD3⁺ CD8⁺ (A) or CD3⁺ CD4⁺ (B) T cells was analyzed by flow cytometry as for Fig. 3C. The percentage of T cells cultured with MV-infected DC (shaded bars) or LV-infected DC (filled bars) that express the molecule minus the percentage of T cells cultured with mock-infected DC that express it is represented. Results are means and SEM from five independent experiments using different donors. Significant differences are indicated as for Fig. 1. The expression of GrzB and perforin in control T cells that had first been stimulated with mock-infected iDC was quantified after restimulation with DC pulsed with inactivated MV (open circles on shaded bars) or with inactivated LV (open circles on filled bars) and is presented as the mean from two independent experiments using different donors. Dot plots showing the expression of CD8 and GrzB or perforin among CD3-gated cells 5 days after the second stimulation with mock-, MV-, or LV-infected iDC are presented. (C) GrzB and FasL 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 target gene/β-actin ratios. Asterisks indicate significant differences between mock-infected and infected cells as for Fig. 1. (D) (Top) ELISPOT assay results showing the number of GrzB spots for 10⁴ T cells first stimulated with MV- or LV-infected iDC or mDC and then restimulated with MV (shaded bars)- or LV (filled bars)-pulsed DC, respectively, minus the number of spots for T cells stimulated with mock-infected DC. Results were obtained with total T cells (iDC and mDC) or with purified CD3⁺ CD4⁺ or CD3⁺ CD8⁺ T cells after the second stimulation and are means and SEM from five independent experiments using different donors. (Bottom) Results of a representative GrzB ELISPOT experiment after stimulation of T cells with mock-stimulated, MV-pulsed, or LV-pulsed iDC. (E) Detection of GrzB by ELISA in supernatants of T cells cultured with mock (open bars)-, MV (shaded bars)-, or LV (filled bars)-infected iDC obtained on day 2 (d2), d5, and d7 of the second and third stimulations. The means and SEM from five independent experiments using different donors are shown. Results are expressed in nanograms per milliliter, and significant differences are indicated as for Fig. 1. (F and G) Titration of MV particles in supernatants of MV-infected iDC (F) or of LV particles in supernatants of LV-infected iDC (G) cultured with total T cells (open symbols) or purified CD3⁺ CD4⁺ (filled symbols) or CD3⁺ CD8⁺ (shaded symbols) T cells that had first been stimulated by mock-infected (circles), MV-infected (squares) (F), or LV-infected (squares) (G) iDC. The means and SEM from four independent experiments using different donors are shown for MV. The means from two independent experiments using different donors are shown for LV. Results are expressed as the number of focus-forming units (FFU) per milliliter.