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

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

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

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

OBJECTIVETo explore a simple, rapid and efficient way to generate dendritic cells from leukemic cells.

METHODSK562 cells were cultured with calcium ionosphere A23187 alone, A23187 plus GM-CSF, or a DC differentiation cocktail consisting of GM-CSF, IL-4 and TNF-alpha, respectively. The expression of surface markers of induced DCs was analyzed by flow cytometry. The K562-DCs stimulating the proliferation of allo-genetic naive T cells and inducing cytotoxicity of T cells were determined by MTT assay.

RESULTSMicroscopic examination revealed that under all the three culture conditions, K562 cells became displaying DC morphology. At 72 hours in the two culture systems containing A23187, there were higher proportions of cells with dendritic morphology [(69.5 +/- 17.2)% and (73.1 +/- 13.9)%, respectively] than that in the cocktail system [(28.5 +/- 12.3)%] (P < 0.05). And the same did when cultured for 7 days [(69.5 +/- 17.2)%, (73.1 +/- 13.9)% respectively vs (51.2 +/- 10.7)%, P < 0.05]. In the 7-day cultures, the percentage of CD1a expressing cells was lower [(8.2 +/- 2.3)% and (10.3 +/- 5.1)% vs (17.2 +/- 1.6)%, respectively] while the CD83 expressing cells was higher [(85.6 +/- 8.8)% and (82.4 +/- 9.1)% vs (77.4 +/- 12.9)%, respectively] compared with that in the cocktail system (P < 0.05). No significant difference was found in the allogeneic T cell proliferation response and induced T cell cytotoxicity between A23187 containing and cocktail groups (P > 0.05).

CONCLUSIONSA23187 treatment is a simple, rapid and efficient in vitro strategy for inducing dendritic cell from leukemic cells.

Calcimycin ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Dendritic Cells ; cytology ; immunology ; metabolism ; Humans ; K562 Cells ; cytology ; drug effects

OBJECTIVETo study the effect of rapamycin in inducing naïve murine effector T cell (Teff) convert to regulatory T cell (Treg) in vitro.

METHODSThe forkhead box protein 3 (FoxP3) negative Teff were isolated and purified from the spleen and lymph node of C57 BL/6 murines aged 6-8 weeks, then Teff were cultured in three groups with mature dendritic cells (mDC), B cells, and plate coated Anti-CD3. In addition, the control wells and the test wells were prepared in each group, rapamycin were not added in the control wells but added in the test wells with concentrations of 1, 10, 50, and 100 nmol/L. Percentages of FoxP3 positive Treg were examined by flow cytometry after 4 days in Anti-CD3 group and after 6 days in the other two groups.

RESULTSAs shown by the flow cytometry, the percentages of FoxP3 positive Treg were as follows in three group: in the mDC group, it was 0.01% in the control well and 0.39%, 0.47%, 0.34%, and 0.26% in test wells; in B cell group, it was 0.01% in the control wells and 5.56%, 5.89%, 7.15%, and 4.72% in the test wells; in Anti-CD3 group, it was 0.93% in the control wells and 1.35%, 1.07%, 1.02%, and 1.19% in test wells. No significant difference was found between the test wells and control wells in the mDC group and Anti-CD3 group; however, the percentages of FoxP3 positive Treg was significantly different between the test wells and control wells in the B cell group (P < 0.01).

CONCLUSIONWhen B cell is acted as the antigen-presenting cell, rapamycin can effectively induce Teff convert to Treg in vitro.

Animals ; B-Lymphocytes ; cytology ; drug effects ; immunology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; immunology ; Flow Cytometry ; Forkhead Transcription Factors ; immunology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred DBA ; Precursor Cells, T-Lymphoid ; cytology ; drug effects ; immunology ; Sirolimus ; pharmacology ; T-Lymphocyte Subsets ; cytology ; drug effects ; immunology ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; 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

Dendritic cells (DC) are now recognized as the most potent professional antigen presenting cells (APC). Several studies on cancer immunotherapy using different approaches to induce cytotoxic T lymphocytes (CTL) in vivo recognizing tumor-associated antigens have been reported. However, the efficacy of immunotherapy in vivo may be limited by the local or systemic suppression of CTL generation or function. To explore the ability of lipopolysaccharide (LPS) stimulated human monocyte-derived DC involved in activity of autologous CD4(+)CD25(+) T cells, HLA-A2 restricted p53(264 - 272) peptide was used as tumor antigen, DC generated with LPS (DC-LPS(+)) or without LPS (DC-LPS(-)) were co-cultured with autologous T cells respectively. The results showed that CD4(+)CD25(+) T cell population in the DC-LPS(+) activated T cells was lower than that in the DC-LPS(-) activated T cells. This finding suggest that the relationship between DC-LPS(+) and population of CD4(+)CD25(+) T cells exists and this property may contribute to regulation of T cell responses to tumor-associated antigens.
CD4-Positive T-Lymphocytes ; cytology ; immunology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Dendritic Cells ; cytology ; immunology ; Humans ; Interleukin-2 Receptor alpha Subunit ; immunology ; Lipopolysaccharides ; pharmacology ; Lymphocyte Activation ; Monocytes ; cytology ; T-Lymphocyte Subsets ; cytology ; immunology

OBJECTIVETo explore the in vitro anti-tumor effect and mechanism of dendritic cell (DC) tumor vaccine induced by astragalus polysacharin (APS).

METHODSPeripheral blood mononuclear cells (PBMCs) isolated from human peripheral blood. DCs obtained from human peripheral blood were cultivated and added with culture solution for in vitro inducing them to immature DCs. On the 5th day of culture, 100 microg/mL (as the final concentration) APS was added to cells in the APS group. DCs were induced to mature in the cytokine groups by adding 20 ng/mL rhTNF-alpha (as the final concentration). Changes of morphology and phenotype of DCs were observed. Mature DCs were sensitized with tumor antigen SGC-7901 and co-cultured with allogeneic T cells. The proliferative function of T lymphocytes was detected by MTT assay. Levels of IL-12 and IFN-gamma in co-cultured supernatant were detected by ELISA. Cytotoxic lymphocytes (CTL) activated by DC were co-cultured with tumor cell SGC-7901. The specific killing capacity of CTL to target cells was detected by LDH release assay.

RESULTSThe morphological observation and phenotypic identification of APS induced DCs were in accordance with the characteristics of mature DCs. APS induced mature DCs could stimulate the proliferation of allogeneic T lymphocytes. The proliferation index of T cells increased with increased ratio of stimulator cells to effector cells (P < 0.05). Levels of IL-12 and IFN-gamma in co-culture supernatant significantly increased in a time-dependent manner (P < 0.05). CTL cells activated by sensitization of DCs could significantly kill tumor cells, and the killing effect increased along with increased effector-to-target ratio.

CONCLUSIONAPS could in vitro induce DCs to mature, promote its antigen-presenting capacity, effectively activate CTLs, and enhance anti-tumor function of the organism.

Antigen-Presenting Cells ; cytology ; drug effects ; immunology ; Cancer Vaccines ; immunology ; Cell Line ; Cell Proliferation ; drug effects ; Coculture Techniques ; Dendritic Cells ; cytology ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Interferon-gamma ; immunology ; Interleukin-12 ; immunology ; Leukocytes, Mononuclear ; cytology ; immunology ; Lymphocyte Activation ; T-Lymphocytes, Cytotoxic ; cytology ; drug effects