Figure 1.

CD30 Targeting Enhances Antigen-Specific Cytolysis by Anti-CEA CAR T Cells

(A–C) Targeting of CD30 by anti-CD30 immunotoxin or anti-CD30 CAR T cells resulted in the depletion of CD30⁺ T cells. Peripheral blood T cells were activated by CD3/CD28 stimulation, and they were incubated for 48 h in the presence or absence of the anti-CD30 immunotoxin Ki4-Eta (1 μg/mL) (A) or T cells engineered with first-generation anti-CD30 and anti-CEA CARs, respectively (B). CD30 expression by T cells in the presence of anti-CD30 immunotoxin (A) or anti-CD30 CAR T cells (B and C) was determined by flow cytometry, and the mean values of CD30⁺ cells of 5 healthy donors in the presence of anti-CD30 or anti-CEA CAR T cells were determined (C). (D and E) Target cell lysis of CEA⁺ tumor cells upon depletion of CD30⁺ lymphocytes. (D) Anti-CEA CAR T cells (2.5 × 10³ anti-CEA CAR T cells/well) were co-cultivated for 48 h with CEA⁺ LS174T or CEA⁻ Colo320 tumor cells (each 5 × 10⁴ cells/well) in the presence of 1 μg/mL anti-CD30 Ki4-Eta immunotoxin. (E) Anti-CD30 (3 × 10³/well) and anti-CEA CAR T cells (7.5 × 10³/well) were co-cultivated with CEA⁺ LS174T or CEA⁻ Colo320 tumor cells (each 5 × 10⁴ cells/well) for 48 h as described above. Viability was determined by the XTT assay and target cell lysis was calculated. Data represent the mean of replicates ± SD. A representative experiment is shown. (F) CD30 targeting by CAR T cells reduces IL-10, but not IFN-γ or IL-2 secretion. Peripheral blood lymphocytes were engineered with first-generation anti-CD30 or anti-CEA CARs, and they were incubated for 48 h in microtiter wells (5 × 10⁴ cells/well, 5 × 10³ CAR T cells/well) that were coated with agonistic anti-CD3 and anti-CD28 mAbs (each 1 μg/mL). Supernatants were recovered and analyzed for IFN-γ, IL-2, and IL-10 secretion by ELISA. Data represent the means of technical replicates of three different healthy donors ± SD. (G) IL-10-secreting cells express high CD30L. T cells were activated as described in the Materials and Methods, cultivated for 72 h, and stimulated for 12 h with anti-CD3 and anti-CD28 mAbs (1 μg/mL each). IL-10 secretion was determined by the IL-10 secretion assay, and cells were additionally stained with anti-CD3, anti-CD30, and anti-CD30L mAbs. Cells were analyzed by flow cytometry and gates set for IL-10⁺ and IL-10⁻ cells. Data represent mean values of 3 healthy donors ± SD. Significant differences were calculated by the Student’s t test.