Detection of transduced CD8⁺ and CD4⁺ T cells in vivo. The scid mice were given intravenous injections of MDA-MB-435-erbB2 tumor cells (5 × 106 cells) at day 0 followed by intravenous injection of transduced T cells at day 5. Lungs were harvested and prepared for immunohistologic analysis 16 days after tumor inoculation. Lung sections from mice that received 1:1 CD4⁺ (5 × 106) and CD8⁺ (5 × 106) scFv-CD28-ζ transduced T cells (A,E,I), unfractionated transduced T cells (107), mouse no. 1 (B-C,F), mouse no. 2 (G,J-K), or 1:1 CD4⁺ (5 × 106) and CD8⁺ (5 × 106) scFv-α-CEA-γ–transduced T cells (D,H,L) were stained with H&E, with anti-CD4 (green), anti-CD8 (red), and anti-CD11b (blue) mAbs (E-H), or with anti-tag (red) and anti-CD11b (green) mAbs (I-L). Representative fields of 5 sections analyzed are shown. Original magnification × 400. (M) Peripheral blood and spleens were harvested from mice at day 100 and used for detection of neomycin phosphotransferase mRNA (∼400 bp). Both peripheral blood (lanes 4 and 5) and spleens (lanes 7 and 8) of 2 representative mice treated with scFv-CD28-ζ–transduced CD4⁺ and CD8⁺ T cells demonstrated the presence of the neomycin phosphotransferase gene. There was no neomycin phosphotransferase detected in either the peripheral blood (lane 3) or spleens (lane 6) of normal scid control mice. As controls, β-actin was detected in all peripheral blood (lane 3) and spleen samples (lane 8) tested. No neomycin phosphotransferase or β-actin was detected in empty DNA controls (lane 2). Lane 1 = 1 kilobase (kb) Plus markers.