Figure 5.
CD86-28z CAR T cells exhibit high potency toward cHL cell lines in vitro. (A) Summary of the composition of anti-CD86 (CD86-28z), anti-CD30 (CD30-28z), and anti-CD19 CAR (CD19-28z) constructs. (B-C) Representative flow cytometric images (B) and quantification (C; individual results and mean ± SEM of 10 different donors) of transduction efficiencies. Transduction efficiency was determined by staining for the extracellular c-Myc tag. (D) Absolute quantification of the molecule count per cell measured with quantitative flow cytometry. Molecule counts/cell of the indicated cell lines were calculated for CD86, CD30, and CD19, respectively. Molecule counts/cell of the isotype control were subtracted from total molecule counts. Depicted are 3 biological replicates. Data are representative of 2 independent experiments. (E-H) CD86-28z (mocha), CD30-28z (light blue), or CD19-28z (gray) CAR T cells were cocultured with the indicated cell lines (from top to bottom: L-428, L-540, KM-H2, Nalm-6, all transduced with fLuc-GFP). (E) Before cocultures, CAR T cells were stained with a Far Red proliferation dye, and antigen-specific proliferation was determined by trace dilution. Cocultures were analyzed by flow cytometry after 7 days. Top row of each color: CAR cocultured with tumor cells. Bottom row of each color: CAR only. Illustrated are representative histograms of in total 3 different donors. (F) Bioluminescence measurement of CAR-mediated lysis of tumor cells. Cell numbers were plated according to the indicated T-cell:tumor cell ratio. Tumor cell killing was determined after 72 hours. Specific lysis was calculated by normalizing to tumor cell-only controls. (G) IFN-γ release into coculture supernatant measured by ELISA. (F-G) Data are mean ± SEM from 3 independent donors. Statistical significance was calculated using 2-way ANOVA with Sidak multiple comparison correction. (H) Representative histograms depicting granzyme B-positive cells after 48 hours of coculture. Granzyme B was measured by intracellular staining after 12 hours of incubation with GolgiStop and GolgiPlug. Illustrated are representative histograms of in total 3 different donors. For all panels: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, P > .05.

CD86-28z CAR T cells exhibit high potency toward cHL cell lines in vitro. (A) Summary of the composition of anti-CD86 (CD86-28z), anti-CD30 (CD30-28z), and anti-CD19 CAR (CD19-28z) constructs. (B-C) Representative flow cytometric images (B) and quantification (C; individual results and mean ± SEM of 10 different donors) of transduction efficiencies. Transduction efficiency was determined by staining for the extracellular c-Myc tag. (D) Absolute quantification of the molecule count per cell measured with quantitative flow cytometry. Molecule counts/cell of the indicated cell lines were calculated for CD86, CD30, and CD19, respectively. Molecule counts/cell of the isotype control were subtracted from total molecule counts. Depicted are 3 biological replicates. Data are representative of 2 independent experiments. (E-H) CD86-28z (mocha), CD30-28z (light blue), or CD19-28z (gray) CAR T cells were cocultured with the indicated cell lines (from top to bottom: L-428, L-540, KM-H2, Nalm-6, all transduced with fLuc-GFP). (E) Before cocultures, CAR T cells were stained with a Far Red proliferation dye, and antigen-specific proliferation was determined by trace dilution. Cocultures were analyzed by flow cytometry after 7 days. Top row of each color: CAR cocultured with tumor cells. Bottom row of each color: CAR only. Illustrated are representative histograms of in total 3 different donors. (F) Bioluminescence measurement of CAR-mediated lysis of tumor cells. Cell numbers were plated according to the indicated T-cell:tumor cell ratio. Tumor cell killing was determined after 72 hours. Specific lysis was calculated by normalizing to tumor cell-only controls. (G) IFN-γ release into coculture supernatant measured by ELISA. (F-G) Data are mean ± SEM from 3 independent donors. Statistical significance was calculated using 2-way ANOVA with Sidak multiple comparison correction. (H) Representative histograms depicting granzyme B-positive cells after 48 hours of coculture. Granzyme B was measured by intracellular staining after 12 hours of incubation with GolgiStop and GolgiPlug. Illustrated are representative histograms of in total 3 different donors. For all panels: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, P > .05.

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