Tim-3–mediated phagocytosis of apoptotic cells is crucial for the cross-presentation by CD8⁺ DCs. (A) Purified CD8⁻CD11c⁺ or CD8⁺CD11c⁺ splenic DCs were pretreated with rIgG (white histograms), RMT3-23 (black histograms), or RMT4-54 (gray histograms) and then cultured with OVA-loaded apoptotic cells. After 2 hours, both DC subsets were purified again to remove dead cells, and then cocultured with OT-I CD8⁺ T cells at the indicated ratio. For the direct presentation, both DC subsets preincubated with OVA_257-264 peptide (1 nM) in the presence of rIgG, RMT3-23, or RMT4-54 were cocultured with OT-I CD8⁺ T cells at a 1:10 (DC/T) ratio. [³H]thymidine (³H-TdR) uptake was measured at 48 to 60 hours. (B) Production of IFN-γ in the culture supernatant at 48 hours after addition of OT-I CD8⁺ T cells was measured by ELISA. Columns represent mean ± SD of triplicates (**P < .01 compared with rIgG). ND indicates not detectable. Similar results were obtained in 3 independent experiments (A,B).