Fig. 6.

Induction of T cell-mediated cytotoxicity by Stx1B-scFv OKT3. Quantification of specific killing activity upon incubation of effector and target cells in the presence of 7 nM or 35.6 nM lectibody. a Gb3⁺ Ramos or b Gb3⁻ Namalwa cells were co-cultured with PBMCs in the effector-to-target (E:T) ratio of 5:1 and purified Stx1B-scFv OKT3 lectibody. After 48 h, about 93% of Gb3-expressing tumor cells were eliminated in presence of 35.6 nM, while Gb3⁻ cells did not show cytotoxicity. c T-cell specific killing was no longer detected when Ramos cells were pre-treated for 72 h with the GSL synthesis inhibitor PPMP. Upon depletion of Gb3 from the cell surface, the lectibody did not induce redirected lysis of tumor cells. Percent viability was calculated relative to the luminescence from an equal number of input control cells and used to calculate percent specific lysis. Results are expressed as a mean ± SD (n = 3) from 3 separate experiments. The experiments were performed with PBMCs derived from at least 3 different donors. Each dot represents data from individual donors. Data of cell proliferation are not shown in the graph. Statistical differences in independent samples were determined with a two-tailed, unpaired t-test for control and treatment groups, at each time point. Tests with a p-value ≤ 0.05 are considered statistically significant and marked with an asterisk (*). p-values ≤ 0.01 are shown as two asterisks (**), and ≤ 0.001 are summarized with three asterisks (***)