Figure 2

Nef-mediated enhancement of replication is highly dependent on the strength of activating stimuli and the level of T cell activation. A. Freshly-isolated PBMC were cultured in the presence of IL-2 (10 U/ml) alone or with either PHA-P (1 μg/ml), PHA-P (2 μg/ml), or α-CD3/CD28 beads (1 bead per cell) for three days. On day 3, cells were stained with α-CD3-FITC and α-CD25-PE or α-CD3-FITC and α-HLA-DR-PE or isotypic controls and analyzed by flow cytometry. %CD25+, %HLA-DR+, and CD25 and HLA-DR median fluorescence intensity of the CD3+ population is shown. Results are typical of three donors. B. Freshly isolated PBMC were infected with an equivalent MOI. Three days post-infection cells were activated with IL-2 (10 U/ml) and either PHA-P (1 μg/ml), PHA-P (2 μg/ml), or α-CD3/CD28 beads (1 bead per cell) for three days. Three days post-activation, the stimulation media was removed and replaced with IL-2 (10 U/ml)-containing media. p24 in culture supernatants was monitored by ELISA.