Figure 4.

Antigen-specific activation of NS3-specific peripheral CD8⁺ cells. Aliquots of heparinized whole blood were incubated with 10 μg/ml HCV peptides NS3–1 (A and C) or NS3-2 (B and D), 1 μg/ml anti-CD28 and 1 μg/ml anti-CD49d (both from Becton Dickinson) at 37°C for 6 hours, with addition of brefeldin A (Sigma) at 10 μg/ml during the last 4 hours, as described (24, 25). The cells were processed and stained with tetramer HCVNS3-2 and antibodies by using a modified procedure (unpublished data; ref. 24). In brief, the cells were stained with tetramer first, followed by lysing red blood cells with FACS Lysing Solution and permeabilyzing white blood cells with FACS Permeabilizing Solution (both from Becton Dickinson). The processed cells were then stained and analyzed as described in the legend of Fig. 3. The number in the upper right quandrant is the percentage of CD69⁺ or IFN-γ⁺ cells of the total tetramer⁺CD8⁺ cells.