Functional anti-CD19–CAR-transduced T cells were detected in mice after adoptive transfer. (A) T cells were cultured and transduced as described in Figure 1C. Two groups of mice received 5 Gy of TBI. After the TBI, the mice were injected intraperitoneally with 38c13 lymphoma cells. The next day, one group of mice received T cells transduced 1D3-28Z.1-3, and the other group received T cells transduced with 1D3-28Z. Both groups received IL-2 on the day of T-cell infusion and the next day. Eight days after the T-cell transfer, the mice were killed and splenocytes were stained with mouse anti–rat Fab antibodies to detect CAR-transduced cells. Splenocytes were also stained with isotype control antibodies. The plots are gated on CD3⁺ cells, and the numbers on the plots represent the percentage of total CD3⁺ cells. The results are representative of 3 experiments. (B) Mean absolute numbers of CAR-expressing CD3⁺CD8⁺ splenocytes in mice that received either 1D3-28Z.1-3–transduced T cells or 1D3-28Z–transduced T cells. Mice were treated as described in Figure 3A. (C) Mean absolute numbers of CAR-expressing CD3⁺CD4⁺ splenocytes in mice that received either 1D3-28Z.1-3–transduced T cells or 1D3-28Z–transduced T cells. Mice were treated as described in Figure 3A. Results are representative of 3 experiments. (D) Three groups of mice received radiation and intraperitoneal injections of 38c13 lymphoma cells as described in Figure 3A. The mice were then injected with 1D3-28Z.1-3–transduced T cells (top row), 1D3-28Z–transduced T cells (middle row), or no T cells (bottom row). Eight days after T-cell transfer, the mice were killed, and splenocytes were cultured with 38c13, CD19-K562, or NGFR-K562. 38c13 and CD19-K562 were CD19⁺. NGFR-K562 was CD19⁻. Intracellular cytokine staining was performed for IFN-γ. The numbers on the plots represent the percentage of CD3⁺ cells. This is 1 of 2 representative experiments.