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. 2016 Jun 28;24(8):1423–1434. doi: 10.1038/mt.2016.82

Figure 1.

Figure 1

Anti-CD30 chimeric antigen receptor (CAR) T cells mediate a specific response against CD30+ lymphoma cells in vitro and in vivo. Anti-CD30 CAR T cells lyse CD30+ lymphoma cells of different origin in a dose-dependent manner. (a) T cells from the peripheral blood were engineered with the anti-CD30 CAR and cocultivated in serial dilutions (0.5–5 × 104/well) with CD30+ MyLa cutaneous T lymphoma cells or CD30– Colo320 tumor cells (5 × 104/well) for 48 hours in 96-well round bottom plates. Coincubation of lymphoma cells with T cells without CAR (w/o CAR) served as control. The assay was done in triplicates and mean values ± standard deviation (SD) were determined. Data represent mean values of one representative out of three experiments. (b) CAR T cells (2.5 × 104/well) were cocultivated with CD30+ MyLa, L1236 Hodgkin's lymphoma cells, and CD30– Colo320 tumor cells (each 5 × 104/well), respectively. T cells without CAR (w/o) served as control. Specific lysis of tumor cells was determined by an XTT-based assay. The assay was done in triplicates and mean values (±SD) were determined. Data represent mean values of one representative out of five experiments. P values were calculated by Student's T-test. Asterisks indicate significant differences between groups (P < 0.05). (c) Anti-CD30 CAR T cells suppress tumor growth in vivo in a dose-dependent fashion. CAR T cells (2.5 × 105–1 × 107 cells/mouse) were coinjected with MyLa cutaneous T lymphoma cells (107/mouse, groups of five or six animals) and tumor growth was monitored every 2–3 days. Mean values of the tumor volume (left) and the area under curve values (right) (±SD) were determined. P values were calculated by Student's T-test. Asterisks indicate significant differences (P < 0.05) compared with the control group. (d) Blocking of CAR T cells by soluble CD30-Fc. MD45 T cells with anti-CD30 CAR were cocultivated with CD30+ L540cy cells (each 5 × 104/well) in presence of increasing amounts of CD30-Fc, the HRS3-specific anti-idiotypic mAb 9G10 and an isotype control mAb, respectively. After 48 hours, culture supernatnants were removed, analyzed for IL-2 secretion and blocking of CAR T-cell inhibition was determined as described in Materials and Methods. The assay was done in triplicate samples and the mean values ± SD were determined.