Figure 1. Sequential LCL stimulation affects EBV-specific T cell development . Samples were taken 7-9 days after each stimulation round (SR) from six different donor CTL lines. (A ) The T cell ratio shows rapid transformation to a highly-enriched CD8+ T cell product by stimulation round 3-4, with most gated CD8+/CD45RO+ cells co-expressing IFN-γ and TNF-α by stimulation round 2, peaking at SR3. Flow plots are from representative CTL line NZ873. The mean percentage of (B ) T cell subpopulations (CD8+, CD4+, CD8-/CD4-) and (C ) CD45RO+, CD45RA+ and CD57+ in CD8 cells at each SR (n=6) is shown, demonstrating consistent development of CD8+ enriched products and consistent differentiation status by SR3. (D ) The mean cMFI of IFN-γ, TNF-α and IL-2 expression in CD8+/CD45RO+ cells reaches a plateau by SR3 then decreases, indicating that peak functionality occurs within 3 stimulation rounds. Data are represented as means ± SEM.