Figure 3
m909-28Z CAR T cells are reactive against endogenous FRβ on human AML cell lines in vitro. To test m909 CAR T-cell reactivity against clinically relevant targets, we acquired 3 human AML cell lines with varying levels of FRβ expression. Cocultures were performed at an E:T ratio of 1:1 unless otherwise noted. Control CD19-28Z CAR T cells are specific for human CD19 and do not express GFP. In media controls, T cells were plated without target cells. Error bars represent mean ± SEM. (A) Surface expression of FRβ on AML cell lines THP1, MV411, and HL60 was determined by flow cytometry using m909-IgG (black) and human IgG isotype control (gray). Percentages represent the proportion of cells with a positive fluorescence signal compared to isotype. (B) Relative FRβ mRNA expression was confirmed using quantitative RT-PCR. Indicated mRNA expression is shown relative to HL60. (C) Antigen-specific IFN-γ (IFNg) secretion was quantified by ELISA after overnight coculture. Each data point represents the mean value of triplicate wells from independent experiments. Represented are n = 10 different normal T-cell donors. (D) m909-28Z CAR T cells proliferate in response to THP1 and MV411, but not HL60, compared to control T cells. PKH26 dilution was measured via flow cytometry before (Pre) and after 5 days in coculture. Overlaying histograms display day-5 PKH26 fluorescence in GFP (gray line), CD19-28Z (dotted black line), and m909-28Z (solid black line) T-cell cocultures with the indicated cell targets. A live, CD3+ gate was used. Percentages represent the proportion of m909-28Z T cells with diluted PKH26 compared to CD19-28Z CAR T cells. (E) m909 CAR T cells exhibit specific lysis of THP1. Luciferase-expressing target cells were cocultured with CAR T cells at an E:T ratio of 1:1. Residual luciferase signal was determined after 24 hours. Percent lysis was determined by luminescence comparison with untreated target wells. Data shown are mean ± SEM of n = 9 independent T-cell donors. P values are calculated compared to CD19-28Z control treated wells. (F) Decreased FRβ expression on THP1 cells surviving overnight coculture with m909 CAR T cells. FRβ surface expression was determined by flow cytometry using m909-IgG and human IgG isotype control. A live, CD3− gate was used to distinguish surviving THP1 cells. The percent of cells showing positive FRβ staining compared to isotype (left) and the FRβ median fluorescence intensity (MFI; right) were determined for triplicate wells (n = 3). P values were determined compared to control GFP T cell–treated wells. **P < .01; ***P < .001.

m909-28Z CAR T cells are reactive against endogenous FRβ on human AML cell lines in vitro. To test m909 CAR T-cell reactivity against clinically relevant targets, we acquired 3 human AML cell lines with varying levels of FRβ expression. Cocultures were performed at an E:T ratio of 1:1 unless otherwise noted. Control CD19-28Z CAR T cells are specific for human CD19 and do not express GFP. In media controls, T cells were plated without target cells. Error bars represent mean ± SEM. (A) Surface expression of FRβ on AML cell lines THP1, MV411, and HL60 was determined by flow cytometry using m909-IgG (black) and human IgG isotype control (gray). Percentages represent the proportion of cells with a positive fluorescence signal compared to isotype. (B) Relative FRβ mRNA expression was confirmed using quantitative RT-PCR. Indicated mRNA expression is shown relative to HL60. (C) Antigen-specific IFN-γ (IFNg) secretion was quantified by ELISA after overnight coculture. Each data point represents the mean value of triplicate wells from independent experiments. Represented are n = 10 different normal T-cell donors. (D) m909-28Z CAR T cells proliferate in response to THP1 and MV411, but not HL60, compared to control T cells. PKH26 dilution was measured via flow cytometry before (Pre) and after 5 days in coculture. Overlaying histograms display day-5 PKH26 fluorescence in GFP (gray line), CD19-28Z (dotted black line), and m909-28Z (solid black line) T-cell cocultures with the indicated cell targets. A live, CD3+ gate was used. Percentages represent the proportion of m909-28Z T cells with diluted PKH26 compared to CD19-28Z CAR T cells. (E) m909 CAR T cells exhibit specific lysis of THP1. Luciferase-expressing target cells were cocultured with CAR T cells at an E:T ratio of 1:1. Residual luciferase signal was determined after 24 hours. Percent lysis was determined by luminescence comparison with untreated target wells. Data shown are mean ± SEM of n = 9 independent T-cell donors. P values are calculated compared to CD19-28Z control treated wells. (F) Decreased FRβ expression on THP1 cells surviving overnight coculture with m909 CAR T cells. FRβ surface expression was determined by flow cytometry using m909-IgG and human IgG isotype control. A live, CD3− gate was used to distinguish surviving THP1 cells. The percent of cells showing positive FRβ staining compared to isotype (left) and the FRβ median fluorescence intensity (MFI; right) were determined for triplicate wells (n = 3). P values were determined compared to control GFP T cell–treated wells. **P < .01; ***P < .001.

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