Tim-3 internalizes apoptotic cells through the FG loop in IgV domain. (A) HEK293T cells transiently expressing Tim-3 were cultured with CFSE-labeled apoptotic cells for 60 minutes at 37°C, and then cells were stained with biotinylated RMT3-23 and Alexa 594–avidin. (B) TAMRA-labeled apoptotic cells were added to HEK293T cells transiently expressing Tim-3-GFP under fluorescence microscope. Phagocytosis and Tim-3-GFP localization were analyzed at the indicated time points. White arrowheads indicate apoptotic cells internalized via Tim-3. White bars indicate 5 μm. (C) Alignment of IgV domain of Tim family molecules. The β-strands of Tim-3 were shown with lines. Mutated residues were indicated by red arrowheads. (D) Positions of mutated residues are indicated on 3-dimensional structure of Tim-3 IgV domain. The color of the protein main-chain is gradually changed along the sequence from blue (N-terminal) to red (C-terminal). (E) HEK293T cells transiently expressing wild-type or mutant Tim-3 were cultured with CFSE-labeled apoptotic cells for 60 minutes at 37°C, and then cells were stained with biotinylated RMT3-23 and PE-avidin. Recognition of apoptotic cells by gated HEK293T cells (R1; Tim-3⁻, R2; Tim-3^low, R3; Tim-3^high) was analyzed by flow cytometry. Similar results were obtained in 2 (A,B) or 3 (E) independent experiments.