Ex vivo ICS of novel specificity and generation of short-term cell lines directed against HCV antigens. All novel responses (detected within peptide matrix but not corresponding to previously known optimal epitopes) were determined to be CD8⁺ T cells by generation of T-cell lines. The response ex vivo, which represented 355 SFC/10⁶ PBMCs via ELISpot (not shown), was detectable by ex vivo ICS staining (A) and was expanded via peptide stimulation in presence of interleukin-2 to 30% of CD8⁺ cells within 14 days in coinfected subject C23-02C (B). All novel responses were verified in the latter manner as the majority fell below the magnitude threshold to be detected by ex vivo ICS.