A Preliminary Study On The Biological Characters And Antitumor Function Of Exosomes Derived From Dendritic Cells

Dendritic cell-derived exosomes are small membrane vesicles which originate from endosomes. They express MHC class I , MHC class II molecules and costimulatory molecules, can induced immunity response of T cells. Exosomes have great prospect in antitumor therapy.To establish a method to isolate exosomes (Dex) from DCs, and to analyse the biological characters and function of exosomes in antitumor immunity, immature DCs (imDCs) from human peripheral blood mononuclear cells were plused with the antigen of K562 tumor cells in our research, then exosomes secreted from imDCs and lipopolysaccharide (LPS) -treated mature DCs (mDCs) were isolated by ultracentrifugation and ultrafiltration. The diameter was detected and the profile of exosomes was determined by electron microscope, the molecules in exosomes were detected by Western blotting. To analyse the effect of exosomes on antitumor immunity, the proliferation, the expression of IFN-γ, up-regulation of CD69 and the cytotoxicity of antigen-specific T cells were measured. Our results reveal that exosomes are small flattened sphere vesicles with a average diameter of 72.3 nm, express CD80, CD86, HLA-DR, FasL, CD54 and MFG-E8 molecules. As compared to immature exosomes, exosomes from mDCs proved expressing more CD80 and less MFG-E8, are more potent to induce antigen-specific T cells proliferation and immunity respond in vitro. With presence of mDC and at the optimum concentration, the value of the A is 0.5 ± 0.01, CD69 is up-regulated and (13.40% ±5.76)% T cells is proliferating, (22.76 ± 2.37)% T cells express IFN-γ, (21.3 ± 8.55)% tumor cells are killed at the effector/target ratio of 10:1.Meanwhile, to confirm the molecular mechanism of the function of exosomes we analyse the effect of exosomes on priming T cells proliferation on condition that there are DCs or not or DCs of different mature states, and the differences in function between allo-exosomes and auto-exosomes. The function of exosomes in immunity was detected after some molecules(CD11a, CD11b, CD11c, CD54, MFG-E8 and CD83)were blocked. At last, using confocal microscopy we observed that DCs embedded exosomes and its variation after the some molecules of exosomes were blocked. We found that both imDex and mDex can not prime T cells without DCs or with imDCs. The exosomes derived from mDCs introduced with different cytokines (LPS, TNF-a, CpG, CD40L) are similar in concentration and different in effect. There are no differences in function between auto-Dex and allo-Dex. Both of them induced T cells proliferation when mDCs is present. The immunity function of Dex depends on its CD11a, CD11b, CD11c, CD54, MFG-E8 and CD83 molecules, the effect of priming T cells is reduced when we blocked these molecules. It is proved by confocal microscopy and FACS that CD54 of Dex plays an important role in targeting and being embedded by DCs.