In vivo antigen expansion of wild-type and gene-optimized TCR-transduced murine splenocytes. Activated B6 splenocytes were transduced with vectors encoding wild-type or gene-optimized pmel-1 TCR. (A) TCR expression was determined by flow cytometry using anti-CD8α, anti-Vβ13, and a pool of anti-Vβ2, anti-Vβ3, anti-Vβ4, anti-Vβ5, anti-Vβ6, anti-Vβ8, anti-Vβ10, and anti-Vβ11. All fluorescence-activated cell sorting (FACS) plots show events that are gated for CD8 expression. The numbers indicate the percentage of CD8⁺ T cells with detectable pmel-1 TCR expression, calculated as follows: %Vβ13⁺Vβpool⁺ cells/%Vβpool⁺ cells × 100%. (B) Detection of TCR expression using anti-CD8 antibodies and D^b-hGp100_25-33 tetramer. The numbers indicate the percentage of tetramer⁺ CD8⁺ cells. (C,D) B6 recipients received splenocytes containing either 1 × 10⁶ pmel-1 TCR transgenic CD8⁺ cells (■, only in panel C), 1.5 × 10⁶ wild-type TCR-transduced CD8⁺ cells (▵), or 1.5 × 10⁶ optimized TCR-transduced CD8⁺ cells (▴). Control mice received 20 × 10⁶ activated, nontransduced cells (□). Mice received an intraperitoneal injection of 1 × 10⁷ pfu rVV-hGp100_(25-33) virus at the day of adoptive transfer (C), or mice received a sublethal dose of total body irradiation one day before adoptive transfer (D). At the indicated time points, peripheral blood was collected and analyzed by flow cytometry using APC anti-CD8α, FITC anti-Vβ13, and PE anti-Vβpool. The percentage of Vβ13⁺Vβpool⁺ CD8⁺ cells of total Vβpool⁺ CD8⁺ cells is plotted. For mice that received pmel-1 TCR transgenic cells, the percentage of Vβ13⁺ CD8⁺ cells is plotted. Error bars represent standard deviations (n = 4).