Tim-3 mediates phagocytosis of apoptotic cells by CD8⁺ DCs. (A) Low-density splenocytes were stained with biotinylated control rIgG2a (thin histograms), RMT1-17, RMT2-14, RMT3-23, or RMT4-54 (thick histograms), followed by PE-avidin, FITC–anti-CD11c mAb, and APC–anti-CD8α mAb; then Tim expression on CD8⁻CD11c⁺ or CD8⁺CD11c⁺ cells was analyzed by flow cytometry. The average of mean fluorescence intensity (MFI) ± SD of triplicates is represented in panel B. (C) Purified splenic CD11c⁺ cells prestained with APC–anti-CD8α mAb were treated with rIgG, RMT3-23, or RMT4-54, and then cultured with TAMRA-labeled apoptotic cells at 37°C. After the indicated time period, cells were stained with FITC–anti-CD11c mAb, and percentage recognition of TAMRA-labeled apoptotic cells by CD8⁻CD11c⁺ or CD8⁺CD11c⁺ cells was quantified by flow cytometry. Columns represent mean ± SD of triplicates in panel D (**P < .01 compared with rIgG). (E) (Left panel) Purified splenic CD11c⁺ cells were stained with Alexa 647–anti-CD8 mAb and Alexa 594–RMT3-23. (Right panel) Purified splenic CD11c⁺ cells prestained with Alexa 647–anti-CD8 mAb were cultured with CFSE-labeled apoptotic cells for 60 minutes at 37°C, and then cells were stained with Alexa 594–RMT3-23. Cells were analyzed by confocal microscopy. White bars indicate 5 μm. Similar results were obtained in 3 (A-D) or 2 (E) independent experiments.