Fig. 5.

Ex vivo testing and the phenotype of MART-1–transgenic CTL after in vivo tumor challenge. (A) Splenocytes from mice injected with transduced CD34 were pooled and stimulated ex vivo with MART-1 peptide (1μg/mL) and irradiated feeders. Five days later the cells were used in a CTL killing assay. (B) After mouse culling, splenocytes were collected and used in a CTL assay without any ex vivo stimulation. Transgenic MART-1 CTL from mice injected with transduced progenitors specifically killed the M202 targets but did not kill the M207⁻ controls. Cell cytotoxicities are at an effector:target ratio of 10:1. “Control” indicates splenocytes collected from mice that were injected with nontransduced hHSC and incubated with the M202 targets. Statistical significance for all of the experiments was determined by a paired Student's t test with significant values set at P < 0.05. (C) Samples from blood and spleens of killed mice were collected and used in a FACS-based phenotyping of CD45⁺CD8⁺CD3⁺MART-1⁺ T cells. Control mice were mice injected with nontransduced autologous hHSC. MART-1–expressing cells displayed higher levels of CD25. (D) From the same animals and cell population as in C, we also examined the maturation state of MART-1 cells. The bar graph below indicates the averages from each group shown in the dot plots above and the statistically significant differences between the treated mice and the controls. Asterisks indicate statistical significance. Statistical analysis of the averages (bar graphs) was determined by a Student's t test with significant values set at P < 0.05 to compare the differences between the control and treated mice. For the dot plots, statistical significance was determined by one-way ANOVA (P < 0.05) comparing the different CD8 T-cell subpopulations.