Figure 3. Necrotic cells contain high–molecular weight factors capable of activating CD8⁺ T cells.

(A) SF from B78-FT cells was fractionated by gel-filtration chromatography. Fractions were washed, adjusted to equal volumes, and added to the cultures of CFSE-labeled OT-I splenocytes in the presence of 20 μg/ml OVA. Proliferation and IFN-γ expression of CD8⁺ T cells were analyzed after 48 hours. Data show 1 representative of 2 independent experiments. (B and C) Purified CD11c⁺ DCs (5 × 10⁴) were precultured with F_9–23 or 20 μg/ml OVA for 4 hours. DCs were then washed, and purified CFSE-labeled OT-I CD8⁺ T cells (1 × 10⁵) were added and incubated for 44 hours in the presence or absence of F_9–23 and/or OVA, as indicated. Proliferation of Ag-specific CD8⁺ T cells was determined. Data are representative of 3 independent experiments. (D) F_9–23 was preincubated with lactacystin (LC) or DMSO, extensively washed, and added to OT-I splenocytes in the presence of 20 μg/ml OVA. Proliferation of CD8⁺ T cells was analyzed after 48 hours. ***P < 0.001, 1-way ANOVA with Dunnett’s multiple-comparison test (C) or with Bonferroni’s paired-comparison test (D).