Figure 4. Antigen specific activation and proliferation of naïve human T cells transduced with engineered-TCRs.

(A) Resting CD4⁺ and CD8⁺ CD45RO⁻CD25⁻ T (T_N) cells were cultured in IL-7-containing medium for 7 days followed by ectopic expression of SL9- or gp100-TCR. 7 days later, cells were then activated with either SL9 or gp100 presented by T2 cells. (B) Upregulation of CD25 from T_N cells expressing SL9-TCR upon activation. Cells collected as indicated in (A) were subject to CD25 staining followed by FACS analysis. The data are representative from three different experiments from multiple donors. (C, D) CD8⁺ and CD4⁺ T_N cells expressing SL9-TCR proliferate and expand via activation through SL9 peptide presented by T2 cells. SL9-TCR-transduced cells as indicated in (A) were labeled with CellTrace Violet and the proliferation was monitored at day 4 post activation and the expansion of T cells was determined at day 4 and day 8 post activation. (E) Induction of CD25 and cytotoxicity from gp100-TCR-transduced CD8⁺ T_N cells at 1st TCR-stimulation and during reactivation. gp100-TCR-overexpressing CD8⁺ T_N cells were co-cultured with T2 cells in the presence of gp100. The frequency of CD25⁺ cells and the cytotoxicity were determined 1 day after 1st activation. Activated cells were kept in culture for an extra 2 weeks and were then restimulated again with gp100 presenting T2 cells (2^nd activation). CD25 levels and cytotoxicity were determined 1 day after reactivation. The data are representative from three different experiments from multiple donors.