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Blood, Vol. 113, Issue 16, 3821-3830, April 16, 2009
Tim-3 mediates phagocytosis of apoptotic cells and cross-presentation
Blood Nakayama et al.
113: 3821
Supplemental materials for: Nakayama et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 63.6 KB)
- Figure S1. Flow cytometric analysis of apoptotic cells (JPG, 67.1 KB)
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(A) Thymocytes were irradiated with the indicated doses of UV, and then cultured at 37°C for 2 h. These cells were then stained with FITC-Annexin V and propidium iodide (PI), and analyzed by flow cytometry. Numbers indicate percent cells in each quadrant. (B) Thymocytes were stained with biotinylated RMT1-17, RMT2-14, RMT3-23, RMT4-54, or RG9-35, followed by PE-avidin (thick histograms). Thin histograms indicate staining with biotinylated control rIgG2a. Similar results were obtained in two independent experiments.
- Figure S2. Tim-3 binds to PS (JPG, 38.8 KB)
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(A) ELISA plates coated with PS, PE, PI, or PC were treated with the indicated doses of Tim-2-Ig, Tim-3-Ig, or Tim-4-Ig. Binding of each Tim-Ig to phospholipids was measured by ELISA. (B) Tim-3-Ig (1.6 µg/ml) and Tim-4-Ig (0.5 µg/ml) were pretreated with the indicated doses of rIgG, RMT3-23, or RMT4-54, then applied onto PS-coated ELISA plate. Binding of each Tim-Ig was estimated by ELISA. Similar results were obtained in three independent experiments (A, B).
- Figure S3. Expression of Tim molecules on peripheral blood cells and splenocytes (JPG, 82.2 KB)
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(A) Peripheral blood cells were stained with biotinylated control rIgG2a (thin histograms), RMT1-17, RMT3-23, or RMT4-54 (thick histograms), followed by PE-avidin, FITC-F4/80, and APC–anti-CD11b mAbs, then each Tim expression on gated cells was analyzed by flow cytometry. (B) Collagenase–digested splenocytes were stained with biotinylated control rIgG2a (thin histograms), RMT1-17, RMT3-23, or RMT4-54 (thick histograms), followed by PE-avidin, FITC-anti-CD11c, and APC–anti-CD11b mAbs, then each Tim expression on gated cells was analyzed by flow cytometry. Similar results were obtained in three independent experiments.
- Figure S4. Cross-presentation of cell-associated antigens by splenic DC subsets (JPG, 24.8 KB)
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Indicated cells (OVA or BSA-loaded apoptotic cells; 5 × 104 per well, purified DC subset; 1 × 104 per well, purified OT-I CD8+ T cells; 1 × 105 per well) were cocultured in 96-well flat-bottomed plate. 3Hthymidine (3H-TdR) uptake was measured at 48–60 h. Similar results were obtained in two independent experiments.
- Figure S5. Blocking effect of anti–Tim-3 mAb on the phagocytosis is not mediated through FcRs (JPG, 70.4 KB)
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(A) Intact RMT3-23 and the F(ab′)2 fragments were applied to SDS-PAGE under reducing conditions, followed by Coomassie staining. (B) HEK293T cells expressing Tim-3 were stained with the indicated dose of control rIgG2a (thin histograms), intact RMT3-23 (thick histograms), or the F(ab′)2 (thick histograms), followed by PE–anti-rat Ig κ light chain. (C) Mice were i.v. injected with the indicated mAb (200 µg each per head), and then 2 h later with CFSE-labeled apoptotic splenocytes (2 × 107 per head). One h later, collagenase-digested splenocytes were harvested, and recognition of CFSE-labeled apoptotic cells by splenic CD11c+ cells was analyzed by flow cytometry. Numbers indicate percent cells in the upper right quadrants. Similar results were obtained in two independent experiments.
- Figure S6. Recognition of apoptotic cells by NIH3T3 cells expressing Tim-3 or Tim-4 (JPG, 58.7 KB)
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(A) NIH3T3 cells stably expressing Tim-3 or Tim-4 were stained with biotinylated RMT3-23 or RMT4-54, respectively, followed by PE-avidin (thick histograms). Thin histograms indicate staining with biotinylated control rIgG2a. (B, C) The indicated NIH3T3 cells were cultured with CFSE-labeled apoptotic cells at 37°C for 60 min, and then analyzed by flow cytometry (B). For fluorescence microscopy, these cells were stained with Hoechst 33342 and rhodamine-labeled phalloidin to visualize nuclei and NIH3T3 cells, respectively (C). Similar results were obtained in three independent experiments.
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