Comparison of the ability of autologous monocytes and immature and mature DCs to stimulate CD4⁺ and CD8⁺ T-cell responses against EBV B-LCL freeze–thaw lysates in IFN-γ Elispot assays.

CD4⁺ and CD8⁺ T cells were directly isolated from the blood of EBV-seropositive healthy donor IP2 and were seeded at 10⁵ cells per well. Autologous monocytes, immature DCs, or mature DCs were not pulsed or were pulsed with lysates derived from autologous EBV B-LCL cells, B-, or T-cell blasts (both 10 kd or larger) as indicated and were added to microwells containing T-cell responders. For maturation, immature DCs were treated on day 6 with TNF-α, IL-1β, IL-6, and PGE₂ for 48 hours (see “Materials and methods”). Mature DC-Is were pulsed with lysate during the 48 hours of maturation from immature DC. Mature DC-IIs were first matured for 48 hours and then pulsed with the lysate for an additonal 48 hours prior to addition to Elispot wells. Resulting spots developed after 20-hour incubation were evaluated and presented as described in Figure 1. Each bar represents the mean spot number of triplicates ± SD with 10⁵ CD4⁺ T lymphocytes or CD8⁺ T lymphocytes initially seeded per well. The data shown are from 1 representative experiment of 5 performed using donors IP1 and IP2.