Tim-3 is crucial for the phagocytosis of apoptotic cells and cross-presentation in vivo. (A) Mice (n = 3 per group) were intravenously injected with the indicated mAb (200 μg each per head), and then 2 hours later with CFSE-labeled apoptotic splenocytes (2 × 10⁷ per head). One hour later, collagenase-digested splenocytes were harvested, and recognition of CFSE-labeled apoptotic cells by splenic CD11c⁺ cells was analyzed by flow cytometry. Numbers indicate percentage of cells in top right quadrants. (B) Collagenase-digested splenocytes were harvested from mice treated as described in panel A at indicated time points, and recognition of apoptotic cells by splenic CD8⁺ DCs was analyzed by flow cytometry. Percentage recognition of CFSE-labeled apoptotic cells by CD8⁺ DCs (percentage CFSE⁺CD8⁺CD11c⁺ cells/percentage CD8⁺CD11c⁺ cells × 100) was calculated. Columns represent mean ± SD of triplicates (*P < .05; **P < .01 compared with rIgG). Similar results were obtained in 3 independent experiments. (C) CFSE-labeled OT-I CD8⁺ T cells (2 × 10⁶ per head) were intravenously transferred into B6 mice (n = 3 per group). The next day, mice were intravenously injected with the indicated mAb (200 μg each per head), and then 2 hours later primed with OVA-loaded apoptotic cells (10⁷ per head). Two days later, whole splenocytes were harvested, and CFSE intensity of CD8⁺Vα2⁺ OT-I cells was analyzed by flow cytometry. Percentage of undivided cells in total OT-I cells (D0 per OT-I in C) was calculated, and mean ± SD of triplicates was shown in panel D (**P < .01 compared with rIgG). Similar results were obtained in 3 independent experiments.