Figure 2.

4-1BB improves long-term CD8 T-cell memory. C57Bl/6 mice were vaccinated with 100 µg of plasmid pOVA twice, two weeks apart. Thirty days later, mice were vaccinated with 2 × 10⁵ pfu of rVV-OVA and received 200 µg of either purified anti-4-1BB mAb or rat IgG i.p. (a) At the indicated number of days later, unfractionated spleen cells were cultured directly ex vivo for 6 hr with brefeldin A, in the presence or absence of 1 µg/ml SIINFEKL peptide, then stained for CD8 and intracellular IFN-γ or (b) TNF-α. (c) The percentage of total spleen cells that were CD8⁺. (d) OVA-specific CD4 T cells were quantified in parallel to SIINFEKL-specific CD8 T cells. Unfractionated spleen cells were cultured directly ex vivo in the presence or absence of 400 µg/ml whole ovalbumin protein for 10 hr, stimulated for an additional 8 hr in the presence of brefeldin A, then stained for CD4 and intracellular IFN-γ. Data shown are the average ± SEM (day 6, n = 6 mice per group; day 60, n = 10 mice per group). A second experiment gave similar results.