Activating CTL precursors to reveal CTL function without skewing the repertoire byin vitro expansion

Detection of the functional CD8+ CTL response usually requires in vitro restimulation. The differences between the CD8+ CTL repertoire in freshly isolated precursor cells and CD8+ CTL after short-term in vitro expansion have been generally assumed to be minimal, but have never been defined experimentally. Using staining with P18-I10/H-2Dd tetramers and monoclonal antibodies (mAb) against Vβ, we show the surprising result that there was significant skewing of the CD8+ CTL repertoire after just 7 days of stimulation. In contrast, we found that overnight incubation of precursor cells with peptide allows the functional assessment of CD8+ CTL (which cannot be detected ex vivo from freshly isolatedcells) without changing the absolute number of antigen-specific CTL as measured by tetramer staining or the repertoire of TCR analyzed with mAb. This study affords a better understanding of the differences between the ex vivo and in vitro stimulated CTL repertoire, and provides an approach to reveal a more faithful representation of the functional in vivo CTL response without skewing of the repertoire of T cells detected.