T cells from CMV-IC-stimulated CTLs secrete Th1 and Tc1 cytokines and lyse CMV-pulsed autologous DC targets. (A) Cytokine secretion. After 4 weeks of CTL culture intracellular (ic) cytokine production (IL-2, IFN-γ, and TNF-α) was analyzed by intracellular fluorescence-activated cell sorting (FACS) staining following restimulation with CMV antigen pulsed (CMV⁺) or not pulsed (CMV^-) DCs in the presence of brefeldin A for 8 hours. Cells in the dot plots represent CD3⁺ T cells gated in the FL-3 channel. The relative size of T-cell subsets within the indicated quadrants is expressed as the percentage of all CD3⁺ T-cell events. Results are representative of 5 independent, consecutive experiments. T cells that stained positive for the indicated fluorescent antibody are depicted in different colors. (B) Cytotoxicity. Responder T cells from CMV-IC-pulsed cultures were harvested on day 30, washed, and recultured in triplicate at graded doses, reflecting the indicated effector-target (E/T) ratios, with 5000 fresh CD34⁺ progenitor-derived immature DCs as targets. DC targets (5000 targets/well) were either pulsed with CMV lysate earlier as indicated or not in controls. Effector cells were added at indicated E/T ratios and cultured at 37°C in 200 μL RPMI 1640 plus 3% PHS. Appropriate controls, including targets alone and responders alone at cell doses representing each E/T ratio, were set up in parallel. Cytotoxicity was measured by fluorometric determination of released LDH in the media after 5 hours of coculture. Specific lysis was calculated after adjusting for target and effector spontaneous LDH release. Percentage specific lysis = [(experimental LDH release) - (target spontaneous LDH release) - (effector spontaneous LDH release)]/[(target total LDH release) - (target spontaneous LDH release)] × 100. Results are representative of 4 independent experiments (mean ± SD).