Figure 3.

Killing by serglycin-bound grB is MPR-dependent. CTL degranulate material was obtained as the supernatant of a CTL culture that was incubated for 4 h at 37°C in the presence (or absence, in the case of spontaneous degranulate) of immobilized anti-CD3 antibody. (A–C) After the indicated treatments, degranulate samples were fractionated over a 100-kDa cutoff filter. Equal volumes of samples were analyzed by immunoblot to detect grB. Data are representative of at least three separate experiments. (A) Time-course. Degranulate was incubated at 37°C in a 5% CO₂ atmosphere for the indicated hours and fractionated at time points. (B) pH effect. To degranulate samples, the following buffers were added to a final concentration of 50 mM: sodium acetate, pH 4.5; MES, pH 5.5; HEPES, pH 7.0; Tris, pH 8.0; boric acid, pH 9.0. Samples were incubated 1 h at 37°C. (C) freeze-thaw effect. Samples were subjected to the indicated number of freeze-thaw cycles. One cycle allowed freezing for at least 30 min at -80°C, followed by rapid thaw in a 37°C water bath. (D–F) Jurkat cells were treated with apoptotic stimuli and inhibitor at 37°C. DNA fragmentation was assessed after 3 h, by TUNEL labeling and flow cytometry analysis. Data are representative of at least three separate experiments. (D) grB, either from purified sources (100 ng/ml) or from supernatants of anti-CD3-stimulated CTL (∼100 ng/ml), was added to cells along with AdV or SLO, in the presence or absence of 20 mM M6P. (E) grB, either from purified sources (500 ng/ml) or from CTL supernatants (untreated, ∼25 ng/ml; anti-CD3-treated, ∼400 ng/ml), was added to cells with or without AdV. (F) Cells were treated with isolated granules at the indicated dilutions. Data represent the mean ± SD of five separate experiments (^* 0.01 < p < 0.05; ^*** 0.001 < p < 0.01).