FIG. 6.

Presentation of SIINFEKL from recombinant bacteria in DC2.4 cells is reduced by the proteasome inhibitor lactacystin and the inhibitor of retrograde transport brefeldin A. We infected 10⁵ DC2.4 in 96-well plates with M. bovis BCG-OVApet or M. smegmatis-OVApet at a multiplicity of infection of 1, 10, 50, or 100 for 2 h at 37°C or pulsed them with SIINFEKL peptide. The remaining extracellular bacteria were killed by a 2-h treatment with 200 μg of amikacin per ml at 37°C. Dendritic cells were then washed in PBS and cocultured with 10⁵ B3Z cells for 16 h at 37°C. Supernatants were assayed for IL-2 by solid-phase ELISA. In some instances dendritic cells were treated with 10 μM lactacystin for 3 h (A) or 1 or 5 μg of brefeldin A per ml for 30 min (B). Dendritic cells were fixed in 1% paraformaldehyde and washed extensively before addition of B3Z (B). A statistically significant decrease in presentation is indicated by * (P < 0.05, Student t test).