In vitro–expanded LLO_91-99–specific CD8 CTLs have an effector phenotype and retain a diverse TCR Vβ repertoire 7–14 days after the second in vitro stimulation with peptide-pulsed APCs. (a) CD8 CTL line stained with mAb for CD8α, CD62L, Thy1.1, CD44, CD25, and H2-K^d tetramers complexed with LLO_91-99. Left panel: Dot plot gated on live CD8 and Thy1.1 T cells. Middle and right panels: Dot plots gated on live CD8 T cells. Numbers in upper quadrants represent percentages of total CD8 T cells. (b) Percentage specific lysis by the LLO_91-99–specific CD8 CTL line in the presence of different concentrations of LLO_91-99 peptide was determined by ⁵¹Cr-release assay using P815 (H2^d) target cells. (c) TNF and IFN-γ production by CD8 CTL line was assessed by standard intracellular cytokine staining following in vitro stimulation. (d) CD8 CTL line was stained for TCR Vβ expression with TCR Vβ–specific antibodies. The percentage of LLO_91-99–specific CD8 CTLs stained with each of the TCR antibodies is indicated. Data are representative of two independent experiments.