Anti-CD19 CARs were expressed by T cells and specifically recognized murine CD19. (A) Diagram of the DNA encoding the 1D3-28Z CAR (ψ, retroviral packaging signal). (B) Diagram of the DNA encoding the 1D3-28Z.1-3 CAR. (C) Splenic T cells were placed in culture with anti-CD3/anti-CD28 beads. The cells were transduced 1 day later with retroviruses encoding 1D3-28Z.1-3 or 1D3-28Z. The transduction was repeated the next day. The third day after culture initiation, the cells were analyzed for CAR expression by staining with anti–rat Fab antibodies (top row) or isotype control antibodies (bottom row). Other cells were cultured and analyzed identically but left untransduced. The plots are gated on CD3⁺ cells, which made up greater than 98% of the total cells in the cultures. The numbers on the plots are the percentages of cells in each quadrant. Data are representative of 5 experiments. (D) T cells from 3 different mice were transduced separately with retroviruses encoding either 1D3-28Z.1-3 or 1D3-28Z. The mean percentages of CAR-expressing CD3⁺ cells that were apoptotic as indicated by staining with annexin V after 7 days of culture are shown. (E) Splenic T cells were cultured and transduced with 1D3-28Z.1-3 or cultured identically but not transduced. (F) The third day after the cultures were initiated, the T cells were stimulated with either the CD19⁺ cell line CD19-K562 or the CD19⁻ cell line NGFR-K562. Intracellular cytokine staining for IFN-γ and IL-2 was carried out. The plots are gated on CD3⁺ cells, and the numbers on the plots are the percentages of cells in each quadrant. Results are representative of 2 experiments.