Figure 4.

ChA6 mAb induce apoptosis in CD4⁺ T cells through the activation of the intrinsic pathway. (A) CD4⁺ T cells were incubated with the indicated concentrations of chA6 mAb. Cells were cultured overnight with or without coated anti-CD3 (0.1 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs. Western blot tests with anti-caspase-3, anti-caspase-8, and anti-caspase-9 mAb were performed. As positive control, Hela cells were treated with staurosporine, 5 μM. Amounts of loaded proteins have been controlled for homogeneity by probing membranes with an anti- β-actin mAb. Weak expression of the cleavage products of caspase-9 in CD4⁺ T cells cultured in medium is considered background apoptosis. Results from one representative donor out of three are shown. (B) CD4⁺ T cells were incubated with chA6 mAb (5 μg/ml) in the presence of 15 μg/ml of cross-linking goat anti–human IgG (F(ab′)₂). Mitochondrial-based death pathways were assessed using 3,3′-dihexyloxacarbocyanine iodide (DiOC₆(3)) accumulation that reflects changes in Δψm in the mitochondria. Results from one representative donor out of three are shown.