1. OncoImmunology, 2012, IF=6.283 Dendritic cell exosomes directly kill tumor cells and activate natural killer cells via TNF superfamily ligands 陈卓

2. www.xianimi.com Background Intercellular communication secreted proteins exosomes nano-vesicles 30 to 100 nm plasma membrane-derived lipid bilayer cytosolic materials DCs TNF/FasL/TRAIL NK cells DC-derived exosomes(DCex) TNF-related apoptosis inducing ligand Induce tumor cells apoptosis MHC-I,MHC-II,CD80,CD86,CD40 membrane-associate proteins

3. www.xianimi.com Background Mice: Female C57BL/6 wild type (WT) Tnf -/- Fasl -/- Tumor cell lines C57BL/6 mouse B16 melanoma MC38 colon adeno-carcinoma DBA/2 mouse KLN205 squamous cell carcinoma Splenocytes NK cells BM cells GM-CSF/IL-4 GM-CSF/IL-4/ LPS iDCs mDCs cell culture media ultracentrifugationiDCex (WT/Tnf -/-/Fasl -/-) mDCex (WT/Tnf -/-/Fasl -/-)

4. www.xianimi.com iDCex (WT) mDCex (WT) TEM Fig. 1A FCM MHC-I/MHC-II/CD80/CD86/CD40/CD14 Fig. 1B Homogenous population 30 to 100 nm Oval-biconcave vesicles Undamaged Not contaminated

5. www.xianimi.com mDCex expressed higher Both iDCex and mDCex

6. iDCex (WT) mDCex (WT) intact iDCex lysed intact mDCex lysed iDCex lysed mDCex ELISA Fig. 2A and Fig.2B TNF/FasL FCM Fig. 2C TNF/FasL/ TRAIL Expression of TNF superfamily ligands on DCex Higher in intact or lysed mDCex TNF more than FasL DCex contain only transmembrane TNF Both transmembrane and soluble FasL

7. Both iDCex and mDCex Antibodies specific for extracellular domains mDCex more intense than iDCex

8. www.xianimi.com B16 melanoma cells + mDCex (WT) + iDCex (WT) MTT 48h Fig. 3 50 µg mDCex (WT)+B16 melanoma cells KLN205 squamous cell carcinoma Mc38 colon carcinoma cells + mDCex (WT) 48h DCex kill tumor cells via TNF superfamily ligands Significant cell death in a dose-dependent and time-dependent

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10. 50 µg mDCex (WT) + Z-VaD-fmk DMSO DNA hypo-staining MTT Fig.4A Fig.4B mDCex (Tnf -/-) mDCex (Fasl -/-) mDCex (WT) B16 melanoma cells + 48h MTT Fig. 4C and Fig.4D 50 µg mDCex (WT) + B16 melanoma cells + IgG control anti-TRAIL antibodies MTT Fig.4E Fig.4F DNA hypo-staining A broad-spectrum caspase inhibitorsuppressed mDCex-induced cytotoxicity DCex mediate potent tumoricidal activity via TNFSFLs Hoechst 33342

11. www.xianimi.com TNF- and FasL-deficient mouse mDCex less cytotoxic

12. www.xianimi.com An anti-TRAIL antibody suppressed the tumoricidal activity of mDCex (WT)

13. 100 µg iDCex (WT) 100 µg mDCex (WT) + aNK cells 24hcell-free supernatant ELISA Fig. 5 Graded amounts mDCex (WT) + aNK cells DCex activate NK cells via TNF mDCex induced higher NK cell activation a dose-dependent manner

14. mDCex (Tnf -/-) +NK cell (WT) Dcex (WT) +NK cell (TNFR1 -/-) Dcex (WT) +NK cell (TNFR2-/-) 24hcell-free supernatants ELISA Fig.6 mDCex (Tnf-/-) —— impaired ability to activate NK cells

15. NK cell (TNFR1 -/- or TNFR2 -/-) ——Unable to respond mDCex (WT)

16. NK cell (WT) alone NK cell (WT) + mDCex (WT) + Isotype,DNTNF,αTNF, αTNFR1,αTNFR2 24hcell-free supernatants ELISA Fig.7 Transmembrane TNF, but not soluble TNF, mediates Dcex-induced activation of NK cells.

17. Similar to DCs, DCex express on their surface TNF, FasL and TRAIL, by which they can trigger caspase activation and apoptosis in tumor cells. DCex activate NK cells and stimulate them to secrete IFNγ upon the interaction of DCex TNF with NK-cell TNF receptors. DCex can mediate essential innate immune functions that were previously ascribed to DCs.

20. 正常情况下，荧光染料 Hoechst 33342 只有少量 能透过细胞膜进入细胞，使其染上低蓝色。但是 当细胞发生凋亡时，它的细胞膜通透性增强，因 此进入凋亡细胞中的 Hoechst 33342 比正常细胞 的多，荧光强度要比正常细胞中要高。 此外，凋亡细胞的染色体 DNA 的结构发生了改变， 从而使该染料能更有效地与 DNA 结合，并且凋亡 细胞膜上的 p- 糖蛋白泵功能受到损伤不能有效地将 Hoechst 33342 排出到细胞外使之在细胞内积累 增加等都使凋亡细胞的蓝色荧光增强。