Figure 2.

Blocking of Enhanced CD30 Expression Improved a CAR T Cell Attack

(A) Simultaneous primary and secondary T cell activations by CD3 and CD28 signaling resulted in enhanced CD30 expression by T cells. T cells from healthy donors (n = 4) were activated by adding the agonistic anti-CD3 and anti-CD28 mAbs (each 100 ng/mL) and further cultivated in the presence of 500 U/mL IL-2. After 7 days, cells were re-stimulated in microtiter plates in the presence or absence of resting peripheral blood lymphocytes (PBLs) (each 5 × 10⁴ cells/well) by solid-phase bound anti-CD3 and anti-CD28 mAbs (coating concentration each 1 μg/mL). For comparison, T cells were co-cultivated without stimulation; resting PBLs of two donors were cultivated in the absence of pre-activated T cells. Data represent mean values ± SD. (B) Blocking of CD30 during primary and secondary T cell stimulations resulted in enhanced target cell lysis by anti-CEA CAR T cells. Anti-CEA CAR T cells and non-modified T cells for control (each 1.25 × 10⁴ cells/well) were co-cultivated with CEA⁺ LS174 tumor cells (2.5 × 10⁴ cells/well) in the presence of freshly isolated autologous PBLs (1.25 × 10⁴ cells/well) in microtiter plates that were coated with 1 μg/mL anti-CD3 and anti-CD28 mAbs. To block CD30 signaling, saturating amounts of the blocking anti-CD30 mAb HRS4 were added. For control, the assay was performed without anti-CD3/CD28 stimulation. Viability of target cells was determined by the XTT assay and cytolysis was calculated. IL-10 and IFN-γ secretion into supernatants was determined by ELISA. A representative experiment of 3 is shown. Data represent mean values ± SD. Significant differences were determined by the Student’s t test.