Figure 2
Figure 2. CD123-specific CAR-expressing T cells lyse CD123-expressing tumor cell lines. (A) Flow cytometric analysis of 293T cells transiently transfected to express CD123 (top, black line) or CD19 (bottom, black line). Parental mock-transduced 293T cells were stained with either anti-CD123 or anti-CD19 antibodies (gray filled, top and bottom) to determine background expression levels. (B) Specific cytotoxicity of CD123-CAR–expressing T cells (26292 and 32716) against 293T cells expressing either CD123 (293T-CD123) or CD19 (293T-CD19) by chromium release assay. Data represent mean values of triplicate wells ± standard deviation (SD). (C) Flow cytometric analysis of CD123 on the AML cell line KG1a, the Epstein-Barr virus–transformed LCL cell line, and the CML cell line K562. Percentage of cells positive for CD123 staining (black line) over isotype controls (gray filled) are indicated in each histogram. (D) Specific cytotoxicity of CD123-CAR T cells (26292 and 32716) against the CD19+CD123+ LCL cell line and the CD19−CD123+ cell line KG1a by chromium release assay. OKT3-expressing LCL (LCL-OKT3) and the CD19− CD123− K562 cell lines were used as positive and negative control cell lines, respectively. Data represent mean values of triplicate wells ± SD. (E) CD123 CAR T cells, or control pair-matched T cells, from 3 healthy donors were cocultured with the indicated cell lines for 24 hours at an E:T of 10:1 and the release of IFN-γ and TNF-α were quantified by Luminex multiplex bead technology. Fold elevation of IFN-γ production against KG1a compared with K562 for 26292 and 32716 were 2.3 and 19.1, respectively. Fold elevation of TNF-α production against KG1a compared with K562 for 26292 and 32716 was 5.5 and 16.5, respectively. (F) Pair-matched carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD19- or CD123-specific T cells were cocultured with the indicated stimulator cell lines for 96 hours at an E:T of 2:1 and analyzed by flow cytometry for CFSE dilution. Unstimulated T cells (filled histograms) were used as baseline T-cell proliferation controls. (G) CFSE-labeled target cells were cocultured with CAR T cells for 5 days in the absence of exogenous cytokines at an E:T ratio of 0.5:1. At the end of the culture, cells were stained using anti-CD3 to distinguish between T cells and CSFE-labeled tumor cells.

CD123-specific CAR-expressing T cells lyse CD123-expressing tumor cell lines. (A) Flow cytometric analysis of 293T cells transiently transfected to express CD123 (top, black line) or CD19 (bottom, black line). Parental mock-transduced 293T cells were stained with either anti-CD123 or anti-CD19 antibodies (gray filled, top and bottom) to determine background expression levels. (B) Specific cytotoxicity of CD123-CAR–expressing T cells (26292 and 32716) against 293T cells expressing either CD123 (293T-CD123) or CD19 (293T-CD19) by chromium release assay. Data represent mean values of triplicate wells ± standard deviation (SD). (C) Flow cytometric analysis of CD123 on the AML cell line KG1a, the Epstein-Barr virus–transformed LCL cell line, and the CML cell line K562. Percentage of cells positive for CD123 staining (black line) over isotype controls (gray filled) are indicated in each histogram. (D) Specific cytotoxicity of CD123-CAR T cells (26292 and 32716) against the CD19+CD123+ LCL cell line and the CD19−CD123+ cell line KG1a by chromium release assay. OKT3-expressing LCL (LCL-OKT3) and the CD19− CD123− K562 cell lines were used as positive and negative control cell lines, respectively. Data represent mean values of triplicate wells ± SD. (E) CD123 CAR T cells, or control pair-matched T cells, from 3 healthy donors were cocultured with the indicated cell lines for 24 hours at an E:T of 10:1 and the release of IFN-γ and TNF-α were quantified by Luminex multiplex bead technology. Fold elevation of IFN-γ production against KG1a compared with K562 for 26292 and 32716 were 2.3 and 19.1, respectively. Fold elevation of TNF-α production against KG1a compared with K562 for 26292 and 32716 was 5.5 and 16.5, respectively. (F) Pair-matched carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD19- or CD123-specific T cells were cocultured with the indicated stimulator cell lines for 96 hours at an E:T of 2:1 and analyzed by flow cytometry for CFSE dilution. Unstimulated T cells (filled histograms) were used as baseline T-cell proliferation controls. (G) CFSE-labeled target cells were cocultured with CAR T cells for 5 days in the absence of exogenous cytokines at an E:T ratio of 0.5:1. At the end of the culture, cells were stained using anti-CD3 to distinguish between T cells and CSFE-labeled tumor cells.

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