OBJECTIVETo establish a method for isolating exosomes from dendritic cells (DC), and to analyse its biological characteristics and function in antitumor immunity.

METHODSImmature DCs (im-DC) from human peripheral blood mononuclear cells were loaded with the antigen of K562 tumor cells, then exosomes were secreted from imDC and lipopolysaccharide (LPS) induced mature DC (mDC). The exosomes from imDC and mDC were isolated separately by ultracentrifugation and ultrafiltration. The exosomes diameter was determined, their profile was observed by electron microscope, and the surface molecules were detected by Western blot. To analyse the effect of exosomes on antitumor immunity, the proliferation, IFN-gamma expression, CD69 up-regulation and cytotoxicity of antigen-specific T cells were measured.

RESULTSExosomes were small flattened sphere vesicles with an average diameter of 72.3 nm and expressed CD80, CD86, HLA-DR, FasL, CD54 and MFG-E8 molecules. As compared to immature exosomes, exosomes from mDC were proved to express more CD80 and less MFG-E8, to be more potent for inducing antigen-specific T cells proliferation and immunity respond in vitro: at its optimum concentration, the absorption value of T cell proliferation test was 0.50 +/- 0.01, CD69 was up-regulated and (13.4 +/- 5.8)% of T cells was in proliferating, (22.8 +/-2.4)% of T cells expressed IFN-gamma, and (21.3 +/-8.6)% of tumor cells were killed.

CONCLUSIONA simple and quick method to isolate and analyse exosomes is established. The exosomes can induce antitumor immunity respond.

Cells, Cultured ; Dendritic Cells ; immunology ; secretion ; Exosomes ; immunology ; Humans ; Lymphocyte Activation ; drug effects ; T-Lymphocytes ; drug effects ; immunology

Dendritic cells(DC) are specialized antigen-presenting cells that prime naive T cells to induce initial immune responses. The immature DC capture and process antigens in the periphery, then emigrate to lymphoid organs. There they complete their maturation by upregulating HLA-I, II molecules, costimulatory molecules (eg. CD80, CD86) and adhesive molecules (eg. CD50, CD54, CD58). More studies showed that in vitro only interferons type I (IFN-alpha, beta) accelerate DC maturation in a dose-dependent manner. The DC induced by IFN type I highly express HLA-A, B, C, HLA-DR, costimulatory molecules and adhesive molecules, and they express enhancing effect of T-cells stimulatory activity in vitro. Progress of research in this field was summarized in this paper.
Cells, Cultured ; Dendritic Cells ; drug effects ; immunology ; physiology ; Humans ; Immunophenotyping ; Interferon Type I ; pharmacology

OBJECTIVETo examine the effect of ginsenoside Rb1 (GRb1) on the immune maturation of monocyte-derived dendritic cells (DCs) induced by oxidized low-density lipoprotein (OX-LDL).

METHODSHuman monocytes purified by CD14+ immuno-magnetic beads were differentiated and induced into immature DCs, which were randomly divided into 6 groups, Group A treated with PBS, Group B treated with OX-LDL, Group C and D treated respectively with GRb1 and ciglitazone, Group E and F were pretreated with the two testing drugs respectively followed by OX-LDL. The immuno-phenotypic expression (CD40, CD1a, and HLA-DR) and endocytosis function of DCs were examined using flow cytometry, the concentration of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) in the culture supernatants were measured with ELISA.

RESULTSCompared with Group B, Group E showed significantly lowered immuno-phenotypic expression of DCs in terms of CD40 (67.4 +/- 1.62 vs. 145.69 +/- 14.86), CD1a (79.64 +/- 3.04 vs. 159.89 +/- 6.09), and HLA-DR (46.43 +/- 2.85 vs. 99.33 +/- 17.11), as well as higher endocytosis level (88.13% +/- 1.06% vs. 25.90% +/- 5.77%, all P < 0.01). Meantime, the serum levels of IL-12 (88.65 +/- 5.59 ng/L vs. 716.69 +/- 36.35 ng/L) and TNF-alpha (133.27 +/- 11.98 ng/L vs. 968.10 +/- 36.42 ng/L) obviously decreased (P < 0.01). The surface molecular expression of DCs and the secretion of inflammatory factors in Group F also obviously decreased, showing insignificant difference from Group E (P > 0.05).

CONCLUSIONGRb1 could obviously inhibit the OX-LDL-induced maturation of DCs, showing similar effects to ciglitazone.

Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; immunology ; Flow Cytometry ; Ginsenosides ; pharmacology ; Humans ; Lipoproteins, LDL ; Monocytes ; cytology ; drug effects ; immunology

OBJECTIVEDendritic cells an hyperinsulinemia are both implicated in the pathogenesis of atherosclerosis. The aim of this study is to explore the effect of high concentration of insulin on the maturation of monocyte-derived dendritic cells (MoDCs) and related signal transduction pathways.

METHODSHuman monocytes were purified (over 98%) using Anti-CD14 micro-beads and cultured for 5 days with DC Cellgro medium containing rhGM-CSF (100 microg/L) and rhIL-4 (20 microg/L). Immature DC were then incubated with insulin of various concentrations (0, 1, 10, 100 nmol/L) for 24 hours in the presence or absence of LY294002 (PI3K inhibitor) or PD98059 (MAPK inhibitor). Immunophenotypic expression of CD86 and CD83 were detected using flow cytometry. Endocytosis function of the MoDCs was evaluated using FITC-Dextran and MoDCs secretion IL-12, IFN-gamma and TNF-alpha were measured by ELISA.

RESULTSInsulin induced significantly higher CD83 and CD86 expressions on MoDCs in a dose-dependent manner. The endocytosis function of MoDCs were significantly inhibited and cytokine secretions of IL-12, IFN-gamma and TNF-alpha significantly increased by 10 nmol/L and 100 nmol/L insulin. These effects could be blocked by the LY294002 and PD98059.

CONCLUSIONHyperinsulinemia contributed to atherosclerosis via stimulating immune maturation of MoDCs via both PI3K and MAPK pathways.

Cell Differentiation ; drug effects ; immunology ; Cells, Cultured ; Cytokines ; metabolism ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Humans ; Insulin ; administration & dosage ; pharmacology ; Monocytes ; cytology ; Phagocytosis ; drug effects ; Signal Transduction

To confirm the mechanism of exosomes as tumor vaccines inducing immunity response, dendritic cells (DCs) were induced from human peripheral blood mononuclear cells, while exosomes were isolated from DC loaded tumor antigen. The effect of exosomes on priming T cell proliferation was analysed under conditions with or without DCs, or DCs at different mature stages. The function of exosomes in immunity was detected through block test after blocking some molecules (CD11a, CD11b, CD11c, CD54, MFG-E8 and CD83). The effect of DCs on embedded exosomes was observed by confocal microscopy, the effect of blocking surface molecules on exosomes on DC-embedding exosomes was assayed by flow cytometry. The results indicated that both exosomes derived from imDC (imDex) and exosomes derived from mDC (mDex) could not prime T cells without DC or with imDC. The exosomes derived from mDC induced with different cytokines (LPS, TNF-alpha, CpG, CD40L) were no significant difference in concentrations but were different in effect. The immunity function of exosomes depended on CD11a, CD11b, CD11c, CD54, MFG-E8 and CD83 molecules, the effect of priming T cells is reduced when these molecules were blocked. Confocal microscopy and FACS assay showed that blocking CD11a and CD54 could inhibit exosome-targeted DC and DC-embedded exosomes. It is concluded that the exosomes target DCs through their surface molecules, therefore results in immune response of T cells.
Antigens, Neoplasm ; immunology ; Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; secretion ; Exosomes ; immunology ; Humans ; K562 Cells ; Lymphocyte Activation ; drug effects ; T-Lymphocytes ; cytology ; immunology

OBJECTIVETo study the inhibitive effects of gastric cancer cell-dendritic cell fusion vaccine on tumor cells of proliferation cycle.

METHODSPeripheral blood mononuclear cells were separated from gastric cancer patients and co-cultured with granulocyte macrophage colony stimulating factors(GM-CSF), interleukin-4(IL-4) and tumor necrosis factor-alpha(TNF-alpha) to generate mature dendritic cells. The dendritic cells and SGC7901 cells were fused by polyethylene glycol, and the pure fusion cells were screened out by selective culture systems. The inhibitive effects of gastric cancer cell-dendritic cell fusion vaccines on tumor cell proliferation cycle in vivo and in vitro were detected by flow cytometry.

RESULTSTreated with the fusion vaccine in vitro, the percentages of G(0)/G(1), S and G(2)/M cells of tumor cells were (76.77+/- 4.38)%, (16.50+/- 2.90)% and (6.73+/- 1.59)% respectively. There were significant differences in the percentages of different cell cycle tumor cells between the tumor cells treated with the fusion vaccine and those co-cultured with dendritic cell or T cells alone(P< 0.01). The proliferative index of the tumor cells treated with the fusion vaccine was 23.34+/- 3.51, significantly lower than those co-cultured with dendritic cell and controls (P< 0.05).

CONCLUSIONSFusion vaccines can affect cell cycle of the tumor cells, thus inhibit tumor cell proliferation and growth.

Cancer Vaccines ; immunology ; pharmacology ; Cell Cycle ; drug effects ; Cell Fusion ; Cell Proliferation ; drug effects ; Dendritic Cells ; immunology ; Humans ; Stomach Neoplasms ; immunology ; Tumor Cells, Cultured

This study was purposed to investigate the effect of bacillus calmette-guerin (BCG) on the expansion of human dendritic cells (DC) from peripheral blood of pediatric patients with leukemia in vitro. The experiment was divi-ded into two groups: the control and the test group, and the latter group was divided into 3 subgroups: BCG (only BCG), GTI (GM-CSF, TNF-α, IL-4) and GTIB (GM-CSF, TNF-α, IL-4 plus BCG). On day 9 of culture the DCs were counted in each groups, the phenotypes of DC were determined by flow cytometry and these DC were stained with Wright-Giemsa for observation and photography under microscopy. The results showed that the test groups all obtained a certain amount of typical DC; the number of DC in the BCG subgroup is lower than that in the GTI and GTIB subgroups (t=4.20; 6.36, p<0.01); there was no significant difference between the GTI and the GTIB subgroups (t=2.25; p>0.05). The rate of CD1a+ in the BCG subgroup was obviously higher than that in the control group (t=3.04, p<0.05), but was lower than that in the GTI and the GTIB subgroups (t=2.79, 6.41, p<0.05), there was no significant difference between the GTI and the GTIB subgroups (t=0.65, p>0.05). The rate of HLA-DR+, CD83+ in the BCG group was higher than that in the control group (t=4.77, 4.15; p<0.05), but lower than that in the GTI and the GTIB subgroups (t=6.65, 3.19; p<0.05). The rate of HLA-DR+, CD83+ in the GTI subgroup was lower than that in the GTIB subgroup (t=5.64, 2.98; p<0.05). It is concluded that BCG not only promotes the proliferation of DC derived from human peripheral blood of leukemia patients in vitro, but also cooperates with rhGM-CSF, rhTNF-α and rhIL-4 in promoting the maturation of DCs.
BCG Vaccine ; immunology ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Child ; Dendritic Cells ; cytology ; drug effects ; Humans ; Leukemia ; immunology ; Mycobacterium bovis ; immunology

OBJECTIVE: To investigate the mechanism. METHOD: We isolate the mouse bone marrow cells and cultured with rGM-CSF and rIL-4 to stimulate bone marrow cells to transfer to immature dendritic cells. And then the immature dendritic cells were costimulated with ILPS and different concentrations of Bimingan Granule. RESULT: MHC II, CD80, CD86 were detected by flow cytometer and TNF-alpha, IL-1beta, IL-6 and IL-10 were quantified by ELISA. CONCLUSION Our results showed that Bimingan Granule may significantly inhibit the differentiation of immature dendritic cells to mature dendritic cells.
Animals ; Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; Mice ; Mice, Inbred C57BL ; Rhinitis, Allergic ; drug therapy ; immunology

This study was to establish the method of purifying heat shock protein GP96 from K562 cells and explore the differentiation and function of human DC influenced by heat shock prolein (HSP). Using ammonium sulfate precipitation, conA-sepharose affinity chromatography and DEAE-sephacel anion exchange chromatography GP96 from K562 cells lysate was isolated and purified. The identification of the purified protein was controlled by Western blot with anti-human GP96 IgG. Human dendritic cell derived from peripheral blood mononuclear cell were cultured with purified GP96. The phenotype changes of DC were analyzed by flow cytometry and MLR was detected by MTT. The results showed that 60-80 microg GP96 was purified successfully from 1 x 10(10) K562 cells. DC stimulated with HSP-GP96 had higher expression rates of CD83, CD86, HLA-DR and lower expression rates of CD1a and had stronger ability to induce T cells proliferation. It is concluded that heat shock protein GP96 can be isolated and purified from K562 cells and could induce maturation of dendritic cell. HSP-DC vaccine show stronger ability to induce T cell proliferation than DC.
Antigens, Neoplasm ; isolation & purification ; pharmacology ; Cancer Vaccines ; immunology ; Cell Differentiation ; drug effects ; Dendritic Cells ; cytology ; drug effects ; immunology ; Humans ; Immunophenotyping ; K562 Cells ; chemistry

This study was aimed to investigate the effect of 1,25(OH)(2) vitamin D(3) [1,25(OH)(2) Vit D(3)] on the differentiation, maturation and function of human dendritic cells (DC) in vitro and its mechanism. Human peripheral blood mononuclear cells were induced to differentiate to DC in vitro. The DC in test group were cultured with 1,25(OH)(2) Vit D(3) 1 nmol/L for 9 d, while the DC in control group were cultured with the equivalent of absolute alcohol. The expression of co-stimulatory molecules on DC were analyzed by flow cytometry. T cell proliferation induced by DC was assessed by MTT method. The expression of indoleamine 2, 3-dioxygenase (IDO) protein was determined by Western blot. The results showed that compared with the control group, the expression of CD80, CD83 and CD86 on DC in test group was significantly down-regulated (P < 0.05), while the CD1a was up-regulated (P < 0.05). The expression rate of CD80, CD83, CD86, CD1a in test group were (40.43 ± 9.83)%, (20.04 ± 4.73)%, (14.45 ± 5.38)%, (58.48 ± 10.72)% respectively, while in control group were (29.36 ± 13.34)%, (35.91 ± 10.19)%, (27.15 ± 11.64)%, (72.20 ± 12.79)% respectively. Compared with the control group, 1,25(OH)(2) Vit D(3)-treated DC exhibited a markedly reduced ability to stimulate allogenic T cell proliferation and up-regulated IDO protein expression.It is concluded that 1,25(OH)(2) Vit D(3) efficiently inhibits the maturation of DC and DC-mediated T cell proliferation, which may be related to the up-regulation of IDO protein expression.
Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cholecalciferol ; pharmacology ; Dendritic Cells ; cytology ; immunology ; Flow Cytometry ; Humans ; Immune Tolerance ; drug effects ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; metabolism