Activated pDCs are potent stimulators of both CD4⁺ and CD8⁺ T cells. Proliferation of T cells was measured by 3H-Thymidine incorporation and depicted as proliferation in counts per minute. (A) Peripheral blood leucocytes (1 × 10⁵) were stimulated for 4 days with either 5 × 10³ or 1 × 10³ allogeneic pDCs. IL-3–pDCs evoke lower T-cell proliferation compared with pDCs activated with either CpG-C or FSME vaccine. The left graph shows the pDC:PBL ratio 1:20 and the right graph 1:100. Data are the mean values ± SEM of 3 independent experiments with different donors. (B) Patient-derived (previously vaccinated with KLH) CD4⁺ T cells (1 × 10⁵) were stimulated with 5 × 10³ KLH loaded (black bars) and unloaded (blank bars) autologous pDCs. We loaded pDCs with KLH using serum that contained KLH specific antibodies. KLH loaded pDCs were highly capable of inducing proliferative KLH-specific recall responses. Data shown are the mean values ± SEM of 1 representative experiment. (C) Cytokine production by autologous T cells after stimulation was analyzed by a cytokine bead array and production is depicted in pg/mL. (D-F) Naive CD45RA⁺CD8⁺ T cells specific for gp100_280-288 were cocultured with autologous pDCs activated through CpG-C or FSME vaccine and loaded with either gp100_280-288 (black bars) or the irrelevant tyrosinase peptide (gray bars). After 16 hours of coculture, antigen-specific activation of CD8⁺ T cells was analyzed by measurement of CD69 (D) surface expression and by secretion of IFNγ in the supernatant (E). Antigen-specific T-cell proliferation was measured after 4 days of culture (F). Data shown are mean values ± SEM of 1 representative experiment of 2 independent experiments performed with different donors.