American Society of Hematology

Figure 2

$Figure 2. Anti-CD19–CAR-expressing T cells produce IL-2 and proliferate in response to CD19. T cells were cultured with anti-CD3/anti-CD28 beads and transduced with retroviruses encoding 1D3-28Z.1-3, 1D3-28Z, or SP6-28Z.1-3. On day 5 of culture, 46% of 1D3-28Z.1-3-transduced cells expressed detectable levels of CAR protein and 19% of 1D3-28Z–transduced cells expressed detectable levels of CAR protein. On day 5 of culture, CAR-transduced cells were used in an IL-2 ELISA and a carboxyfluorescein succinimidyl ester (CFSE) proliferation assay. (A) 1D3-28Z.1-3–transduced T cells, 1D3-28Z–transduced T cells, or SP6-28Z.1-3–transduced T cells were cultured overnight alone or with the indicated target cells, and an IL-2 ELISA was performed. The 38c13 and CD19-K562 cells were CD19+ and NGFR-K562 cells were CD19−. The units are in picograms per milliliter. Error bars represent the SEM of duplicate wells. 1D3-28Z.1-3–transduced T cells and 1D3-28Z–transduced T cells produced IL-2 in response to CD19. Minimal IL-2 production occurred when T cells were cultured alone or with NGFR-K562 cells. This is 1 of 2 experiments with similar results. (B) CFSE-labeled 1D3-28Z.1-3–transduced T cells were cultured for 4 days with either irradiated CD19-K562 cells or irradiated NGFR-K562 cells as indicated above each plot. The cells were stained with anti-Fab antibodies to detect the CAR. Almost all 1D3-28Z.1-3–expressing T cells that were cultured with CD19-K562 cells proliferated as indicated by CFSE dilution. Only a minority of 1D3-28Z.1-3–expressing T cells that were cultured with NGFR-K562 cells proliferated. The plots are gated on CD3+ lymphocytes, and the numbers on the plots are the percentages of cells in each quadrant. Results are representative of 2 experiments. (C) Only a small fraction of T cells expressed 1D3-28Z after 4 days of culture with either CD19-K562 cells or NGFR-K562 cells. More of the persisting cells underwent proliferation when they were cultured with CD19-K562 cells than when they were cultured with NGFR-K562 cells. The plots are gated on CD3+ lymphocytes, and the numbers on the plots are the percentages of cells in each quadrant.$

Anti-CD19–CAR-expressing T cells produce IL-2 and proliferate in response to CD19. T cells were cultured with anti-CD3/anti-CD28 beads and transduced with retroviruses encoding 1D3-28Z.1-3, 1D3-28Z, or SP6-28Z.1-3. On day 5 of culture, 46% of 1D3-28Z.1-3-transduced cells expressed detectable levels of CAR protein and 19% of 1D3-28Z–transduced cells expressed detectable levels of CAR protein. On day 5 of culture, CAR-transduced cells were used in an IL-2 ELISA and a carboxyfluorescein succinimidyl ester (CFSE) proliferation assay. (A) 1D3-28Z.1-3–transduced T cells, 1D3-28Z–transduced T cells, or SP6-28Z.1-3–transduced T cells were cultured overnight alone or with the indicated target cells, and an IL-2 ELISA was performed. The 38c13 and CD19-K562 cells were CD19⁺ and NGFR-K562 cells were CD19⁻. The units are in picograms per milliliter. Error bars represent the SEM of duplicate wells. 1D3-28Z.1-3–transduced T cells and 1D3-28Z–transduced T cells produced IL-2 in response to CD19. Minimal IL-2 production occurred when T cells were cultured alone or with NGFR-K562 cells. This is 1 of 2 experiments with similar results. (B) CFSE-labeled 1D3-28Z.1-3–transduced T cells were cultured for 4 days with either irradiated CD19-K562 cells or irradiated NGFR-K562 cells as indicated above each plot. The cells were stained with anti-Fab antibodies to detect the CAR. Almost all 1D3-28Z.1-3–expressing T cells that were cultured with CD19-K562 cells proliferated as indicated by CFSE dilution. Only a minority of 1D3-28Z.1-3–expressing T cells that were cultured with NGFR-K562 cells proliferated. The plots are gated on CD3⁺ lymphocytes, and the numbers on the plots are the percentages of cells in each quadrant. Results are representative of 2 experiments. (C) Only a small fraction of T cells expressed 1D3-28Z after 4 days of culture with either CD19-K562 cells or NGFR-K562 cells. More of the persisting cells underwent proliferation when they were cultured with CD19-K562 cells than when they were cultured with NGFR-K562 cells. The plots are gated on CD3⁺ lymphocytes, and the numbers on the plots are the percentages of cells in each quadrant.

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