Bacillus Calmette-Guerin (BCG) is reported to suppress Th2 response and asthmatic reaction. Dendritic cells (DCs), the major antigen-presenting cells, infections with BCG are known to result in inducing various cytokines. Thus, DCs are likely to play a role in the effects of BCG on asthma. This study aims at investigating that cytokine milieu secreted by BCG-treated DCs directly enhances allergen-specific Th1 response and/or suppresses Th2 response in allergic asthma. DCs and CD3+ T cells were generated from Dermatophagoides farinae-sensitive asthmatics. DCs were cultured with and without BCG and subjected to flow cytometric analysis. IL-12 and IL-10 were determined from the culture supernatants. Some DCs were cocultured with T cells in the presence of D. farinae extracts after adding the culture supernatants from BCG-treated DCs, and IL-5 and IFN-gamma were determined. BCG-treated DCs enhanced significantly the expressions of CD80, CD86, and CD40, and the productions of IL-12 and IL-10. Addition of culture supernatants from BCG-treated DCs up-regulated production of IFN-gamma by T cells stimulated by DCs and D. farinae extracts (p<0.05), but did not down-regulate production of IL-5 (p>0.05). The cytokine milieu secreted by BCG-treated DCs directly enhanced allergen-specific Th1 response, although did not suppress Th2 response.
Antigens, Dermatophagoides/*immunology ; Asthma/*immunology ; Cells, Cultured ; Coculture Techniques ; Culture Media ; Cytokines/*immunology/secretion ; Dendritic Cells/cytology/*immunology/secretion ; Humans ; Hypersensitivity/immunology ; Interferon Type II/immunology/secretion ; Interleukin-10/immunology/secretion ; Interleukin-12/immunology/secretion ; Interleukin-5/immunology/secretion ; Lymphocyte Activation/immunology ; Mycobacterium bovis/*immunology ; Research Support, Non-U.S. Gov't ; Th2 Cells/cytology/immunology/secretion ; Up-Regulation/immunology

The objective of this study was to investigate the effect of dendritic cells (DCs) on expansion and function of autologous natural killer (NK) cells and its mechanism in vitro. NK cells were expanded from peripheral blood mononuclear cells (PBMNCs) of healthy volunteers in stem cell growth medium (SCGM) supplemented with rhIL-2 (control group) in 24-well culture plates at 37 degrees C in a humidified CO(2)-containing atmosphere. NK cells were cultured with autologous DCs in the ratio of 5 to 1 (group 5:1) or 1 to 1 (group 1:1) from day 10 after expansion. Total cells of every group were counted and the expression of CD3, CD16/56 on the surface of NK cells was assayed by flow cytometry on days 7, 14 and 21 to calculate the expansion of NK cells. Cytotoxicity of expanded NK cells against K562 cells was assayed by MTT method. TNF-alpha and IL-12p70 were detected in culture supernatants by sandwich ELISA. The results indicated that the expansion and cytotoxicity of NK cells were improved after mixed with autologous DCs. Furthermore, when DCs were mixed with NK cells, the ratio of DCs to NK cells was higher, the expansion and cytotoxicity NK cells were higher. On day 14, the expansion multiple in control, group 5:1 and group 1:1 were 16.26 +/- 1.58, 29.25 +/- 4.01 and 21.23 +/- 2.91 respectively. The expansion multiple of group 5:1 was much higher than that of the other two groups (p < 0.05). The expressions of CD3(-), CD56/16(+) on surface of NK cells in control, group 5:1, group 1:1 were (34.8 +/- 5.1)%, (64.6 +/- 7.8)% and (50.6 +/- 8.7)% respectively and that of group 5:1 was the highest (p < 0.05). The cytotoxicities against K562 cells in control, group 5:1 and group 1:1 were (63.7 +/- 3.8)%, (87.4 +/- 6.8)% and (75.4 +/- 6.3)% respectively. The cytotoxicity of group 5:1 was higher than that in the other two groups also (p < 0.05). TNF-alpha and IL-12p70 levels in culture supernatants when DCs and NK cells were mixed in the ratio of 5 to 1 were much higher than those in culture supernatants of DCs and NK cells alone or in culture supernatants when DCs and NK cells were mixed in the ratio of 1 to 1 (p < 0.05). It is concluded that the expansion and cytotoxicity of NK cells can be improved by DCs and it depended on the mixed ratio of DCs to NK cells. The elevated expansion of NK cells by DCs bears relation to IL-12 produced by DCs. The enhanced cytotoxicity of NK cells is associated with TNF-alpha secreted by NK cells.
Cells, Cultured ; Coculture Techniques ; Dendritic Cells ; cytology ; immunology ; Humans ; Interleukin-2 ; secretion ; Killer Cells, Natural ; cytology ; immunology ; Tumor Necrosis Factor-alpha ; secretion

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

Cell mediated immune responses play a prominent role in syphilis, which is caused by Treponema pallidum. The role of dendritic cells (DC) in the syphilitic infection is not well understood in human. In the present study, we studied interaction of T. pallidum with DC, generated from human peripheral blood mononuclear cells with GM-CSF and IL-4. After adding T. pallidum for 16 hours to immature DC at culture day 7, the change of surface antigens on DC was monitored by flow cytometry, the amount of IL-12 in culture supernatant of DC was measured by ELISA and T cell stimulatory capacity of DC was checked in mixed lymphocyte reaction (MLR). We have observed an efficient phagocytosis of T. pallidum by electron microscopy as early as 2 hours after addition of T. pallidum to DC. Interaction of DC with T. pallidum resulted in increased surface expression of CD83 which was proportionally increased according to the number of T. pallidum. Expressions of CD80, CD86 and HLA-DR on DC were slightly increased. The amount of IL-12 in the culture supernatant of DC was increased (1, 099pg/ml) after the addition of T. pallidum. T. pallidum-infected DC also displayed enhanced T cell stimulatory capacity in MLR. As seen from the above, we observed phagocytosis of T. pallidum by DC as early as 2 hours after addition of T. pallidum to DC and found that T. pallidum can stimulate DC maturation which mean that DC modulate an protective immune response during T. pallidum infection.
Cells, Cultured ; Dendritic Cells/cytology/*immunology/*microbiology ; Human ; Interleukin-12/metabolism/secretion ; Lymphocyte Culture Test, Mixed ; Microscopy, Electron ; Phagocytosis/immunology ; Receptors, Cell Surface/immunology/metabolism ; Support, Non-U.S. Gov't ; Syphilis/*immunology ; T-Lymphocytes/immunology ; Treponema pallidum/*immunology/ultrastructure

OBJECTIVETo investigate the effects of IL-2 gene modification enhancement of the antigen-presenting function of the mouse bone marrow derived dendritic cells and on the activation of CTL induced by MHC class I molecule restricted antigen peptides as well as the related immunological mechanisms.

METHODSDCs were prepared from mouse bone marrow and modified by recombinant IL-2 adenovirus (DC-IL-2). The IL-12 and IFN-gamma levels in culture supernatant of DC and CTL were examined by ELISA, the expression of costimulatory molecules and fluorescent intensity of endocytosis of OVA-FITC in DC by FACS, the capacity of presenting 3LL cell tumor antigen by (3)H-TdR incorporation method, the MHC class I-restricted tumor-antigen-peptide Mut1 of 3LL cells pulsed DC-IL-2 to induce CTL cytotoxicity by (51)Cr 4-hr releasing assay.

RESULTSAfter IL-2 gene modification, DC-IL-2 could produce high level of IL-12 [(78.4 +/- 6.6) pg.(1 x 10(6) cells)(-1).ml(-1)]. The expression of costimulatory molecules on DC-IL-2 was increased, the fluorescent intensity of DC captured OVA-FITC was enhanced, and the proliferation of allo-T cells from 3LL bearing mouse pulsed with Mut1 was also enhanced. Mut1 antigen peptide pulsed DC-IL-2 could induce more potent antigen-specific CTL cytotoxicity and excrete high concentration of IFN-gamma [(1 168 +/- 58.4) pg/ml] in vivo.

CONCLUSIONIL-2 gene modification of DC can activate second signal for DC presenting antigen, and enhance the function for capturing and presenting tumor antigen. DC-IL-2 pulsed with MHC class I restricted tumor-antigen-peptide can induce specific anti-tumor immune response more effectively. Owing to IL-2 gene modification, the functions of IL-12 excretion and T cell activation of DC were promoted, so that the capacity of CTL excreting IFN-gamma was enhanced, which are relevant to the immune mechanism.

Adenoviridae ; genetics ; Animals ; Antigen Presentation ; immunology ; B7-1 Antigen ; genetics ; metabolism ; Dendritic Cells ; cytology ; immunology ; Female ; Interleukin-12 ; secretion ; Interleukin-2 ; genetics ; Lymphocyte Activation ; genetics ; immunology ; Mice ; Mice, Inbred C57BL ; Recombination, Genetic ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; cytology ; immunology

Atherosclerosis ; etiology ; prevention & control ; Cytokines ; secretion ; Dendritic Cells ; drug effects ; immunology ; Endocytosis ; Fenofibrate ; pharmacology ; Humans ; Immunophenotyping ; Lipoproteins, LDL ; toxicity ; Monocytes ; cytology ; PPAR alpha ; agonists ; physiology

OBJECTIVETo explore the changes of surface antigen and function of rituximab on dendritic cells derived from patients with Primary immune thrombocytopenia (ITP) to further understand the effective mechanism of immunotherapy.

METHODSThe peripheral blood mononuclear cells (PBMCs) were isolated from remission patients with ITP before and after low-dose rituximab infusion, and the PMNCs were stimulated for 5 days by rhGM-CSF and rhlL-4 in 5% CO2 air at 37°C incubator. Then all of DCs were cultured with TNF-α for 48 hours. The morphology of DCs was monitored under inverted microscope daily, and the surface antigens of the DCs were analysed by flow cytometry, meanwhile the levels of IL-12p70 and TGF-β1 in supernatants were detected by ELISA, mix lymphocyte reaction was performed by MTT assay.

RESULTS(1) Rituximab-treated-DCs showed no obvious tree-like protruding compared with untreated-DCs. The former cells were small and most of nucleus were centric. (2) The expressions of HLA-DR, CD80, CD83 and CD86 on rituximab-treated-DCs \[56.37 ± 3.95)%, (36.41 ± 2.82)%, (30.45 ± 4.61)% and (41.98 ± 4.17)%, respectively\] were significantly lower than those untreated-DCs \[(73.71 ± 7.61)%, (55.14 ± 7.30)%, (80.91 ± 7.09)% and (59.03 ± 3.43)%, respectively\](all P < 0.05), the concentration of IL-12p70 was significantly lower, \[(66.87 ± 4.29)% vs (50.17 ± 14.52)%\], while that of TGF-β1 \[(9.70 ± 0.31)%\] higher than the untreated-DCs \[(2.70 ± 0.36)%\] (P < 0.05). (3) The abilities to activate T cells proliferation of rituximab-treated-DCs reduced compared with untreated-DCs.

CONCLUSIONThe surface antigen of ITP-DCs and the concentration of IL-12p70 reduced after the low-dose rituximab infusion. The abilities to activate T cells proliferation reduced while the concentration of TGF-β1 increased. Rituximab may achieve its therapeutic effect on ITP by downregulating the immunoreactivity of DCs.

Antibodies, Monoclonal, Murine-Derived ; administration & dosage ; therapeutic use ; Cell Proliferation ; Cells, Cultured ; Dendritic Cells ; cytology ; metabolism ; secretion ; Female ; Humans ; Interleukin-12 ; metabolism ; Lymphocyte Activation ; Male ; Rituximab ; T-Lymphocytes ; immunology ; Thrombocytopenia ; drug therapy ; immunology ; metabolism ; Transforming Growth Factor beta1 ; metabolism

BACKGROUND AND OBJECTIVECytokine-induced killer (CIK) cells and autologous dendritic cells-CIK (DC-CIK) cells co-cultured with autologous dendritic cells (DCs) and CIK cells are commonly used for immunotherapy recently. We compared the anti-tumor immune response of CIK cells, autologous DC-CIK cells, and semi-allogeneic DC-CIK cells to explore a more effective anti-tumor adoptive immunotherapy approach.

METHODSPeripheral monocytes were isolated from patients with renal carcinoma, lung cancer, or maxillary squamous cell carcinoma and their healthy adult children. Isolated cells were cultured and induced as DCs and CIK cells in vitro. CIK cells from patients were co-cultured with autologous DCs and DCs from their children respectively, generating DC-CIK cells and semi-allogeneic DC-CIK cells. The anti-tumor activities of autologous CIK cells, autologous DC-CIK cells, and semi-allogeneic DC-CIK cells were measured by LDH assay. Intracellular staining was used to test the secretion of cytokines. Flow cytometry was applied for detecting the phonotype changes of these three types of cells. Cell proliferation and cell apoptosis were detected by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and Annexin V/PI respectively.

RESULTSCompared with autologous CIK cells and DC-CIK cells, semi-allogeneic DC-CIK cells significantly enhanced the anti-tumor activity and IFN-gamma secretion, reduced IL-4 secretion, increased the ratio of CD3(+)CD56(+) cells and CD3(+)CD8(+) cells, decreased the number of CD4(+)CD25(+) cells, promoted cell proliferation, and lessened cell apoptosis.

CONCLUSIONSSemi-allogeneic DC-CIK cells had a stronger anti-tumor effect than did autologous CIK cells and DC-CIK cells. Our results provided experimental evidence for clinical application of DC-CIK cells.

Apoptosis ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Cytokine-Induced Killer Cells ; cytology ; immunology ; metabolism ; Cytokines ; metabolism ; Cytotoxicity, Immunologic ; Dendritic Cells ; cytology ; immunology ; metabolism ; Hep G2 Cells ; Humans ; Immunotherapy, Adoptive ; Interferon-gamma ; secretion ; Interleukin-4 ; secretion ; K562 Cells ; Kidney Neoplasms ; metabolism ; pathology ; L-Lactate Dehydrogenase ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; Maxillary Neoplasms ; metabolism ; pathology

OBJECTIVETo investigate if granulocyte-macrophage colony stimulating factor (GM-CSF) gene-modified dendritic cells (DC) enhance antitumor immunity in vitro.

METHODSMice were injected with chemokine ligand 3 (CCL3) via the tail vein. Fresh B220(-)CD11c(+) cells were sorted from the peripheral blood mononuclear cells (PBMCs) and cultured into DCs by cytokines.DCs were transfected with AdGM-CSF gene at different ratios of multiplicity of infection (MOI) to determine the optimal gene transfection conditions, and the expression of GM-CSF was detected after transfection. The variation of GM-CSF gene-modifiedDCs were analyzed by morphological examination, phenotype analysis, and mixed lymphocyte reaction (MLR).DCs were loaded with gastric cancer antigen obtained by freezing and thawing method. The killing effect of DCs vaccine-stimulated T lymphocytes on gastric cancer cells was assessed by MTT assay. INF-gamma production was determined with the INF-gamma ELISA kit.

RESULTSB220(-)CD11c(+) cells increased obviously after CCL3 injection. The ELISA results showed that after GM-CSF gene modification, DCs could produce high level of GM-CSF. When DCs were transfected with AdGM-CSF gene at MOI equal to 100, the GM-CSF level in culture supernatants reached saturation [(130.00 +/- 12.61) pg/ml]. After GM-CSF gene-modification, DCs tend to be more maturated as detected by morphological observation and phenotype analysis. At the same time, the capacity of activating the proliferation of allogeneic T lymphocytes was enhanced greatly. T lymphocytes stimulated by DCs transfected with GM-CSF gene showed a specific killing effect on gastric carcinoma cells and produced high level of INF-gamma [(1245.00 +/- 13.75) pg/ml].

CONCLUSIONAfter GM-CSF gene modification, DCs can produce high level of GM-CSF, which tend to be more maturated, and the capacity of activating the proliferation of allogeneic T lymphocytes is enhanced greatly. GM-CSF gene modified DCs can induce specific CTL to target tumor cells in vitro.

Adenoviridae ; genetics ; Animals ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; CD40 Antigens ; metabolism ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Cell Proliferation ; Cytotoxicity, Immunologic ; Dendritic Cells ; cytology ; immunology ; metabolism ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; metabolism ; Histocompatibility Antigens Class II ; metabolism ; Interferon-gamma ; secretion ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; Stomach Neoplasms ; immunology ; metabolism ; pathology ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection

OBJECTIVETo explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells (BMDCs) and T lymphocytes in vitro.

METHODSAfter BMDCs stimulated by E.coli LLO/OVA, their Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) receptor signalling pathway were examined by superarray hybridization; and the priming effect of the vaccine activated BMDCs on CD4(+)T and CD8(+)T was determined by [3H]thymidine uptake and ELISA, the tumor cytotoxic effect of activated CD8(+)T cells was determined by cytotoxic assay.

RESULTSAfter BMDCs were activated by E. coli LLO/OVA via TLR4, NOD1 receptor and NF-κB signalling pathway, the expression of their surface molecules including MHC class I, MHC class II, CD40, CD80 and CD86 significantly up-regulated; the secretion of IL-12 and IFN-γ increased also. The mature BMDCs stimulated the allergic CD4(+)T and CD8(+)T cells proliferation and their IL-2 and IFN-γ secretion, and the activated CD8(+)T cells effectively killed B16-OVA melanoma cells and RMA-S/OVA lymphoma cells in vitro.

CONCLUSIONE.coli LLO/OVA is effective in inducing BMDCs maturation via activating TLR4 and NOD1 receptor signalling pathway and promoting specific anti-tumor T cell immunity in vitro.

Animals ; Antigens, Neoplasm ; genetics ; pharmacology ; Bacterial Toxins ; genetics ; pharmacology ; Cancer Vaccines ; genetics ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; immunology ; Coculture Techniques ; Cytokines ; immunology ; secretion ; Dendritic Cells ; cytology ; drug effects ; immunology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Female ; Flow Cytometry ; Heat-Shock Proteins ; genetics ; pharmacology ; Hemolysin Proteins ; genetics ; pharmacology ; Immunity, Innate ; drug effects ; Mice ; Mice, Inbred C57BL ; Nod1 Signaling Adaptor Protein ; genetics ; physiology ; Ovalbumin ; genetics ; pharmacology ; Recombinant Fusion Proteins ; genetics ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology ; Toll-Like Receptor 4 ; genetics ; physiology