The purpose of this study was to explore the effect of NOD2 signalling pathway activated by muramyl dipeptide (MDP) on the immunomodulation effect of human monocyte-derived dendritic cells (DC) loaded with leukemia cell lysates. Peripheral blood mononuclear cells (PBMNC) were isolated by density gradient centrifugation, These cells were cultured with three cytokines for 7 days to induce their maturation. On the 5th day, cells were loaded with leukemia cell HL-60 lysates. NOD2 expression was detected by RT-PCR and Western blot. The phenotype of DC were analyzed by flow cytometry, and ELISA was used to assay levels of IL-12 (p40) . The results showed that MDP could trigger NOD2 mRNA and protein expression in different groups of DC, especially in sensitized DC+MDP group, which was significantly higher than that in the DC+MDP group and sensitized DC without MDP stimulation, the difference was statistically significant (P < 0.05). Besides, the expression of surface molecules (HLA-DR, CD80, CD83, CD86, CD40) in the group of DC loaded with leukemia cell lysate and stimulated by MDP (sensitized DC+MDP) reached the highest level, followed by the group of DC loaded with leukemia cell lysate without MDP and DC only stimulated by MDP, non-treated DC were the lowest (P < 0.05). Similarly, compared with untreated unstimulated DC, after loading with HL-60 lysates or only stimulating with MDP, the secretion of IL-12p40 increased, but IL-12p40 level (573.86 ± 32.09 pg/ml) in DC+MDP group was higher than that in group of sensitized DC (365.03 ± 28.86 pg/ml) (P < 0.05), and it in sensitized DC+MDP group reached the highest (898.30 ± 61.08) pg/ml, compared to other groups (P < 0.05). It is concluded that MDP can significantly enhance the NOD2 mRNA and protein expression in sensitized DC, promote the expression of HLA-DR, synergistic costimulatory molecules and adhesion molecules of DC, at the same time, MDP can increase secretion of inflammatory factors IL-12p40. This study will provide a new ideas for DC application in leukemia immunotherapy.
Acetylmuramyl-Alanyl-Isoglutamine ; pharmacology ; Cells, Cultured ; Dendritic Cells ; immunology ; metabolism ; HL-60 Cells ; Humans ; Interleukin-12 Subunit p40 ; secretion ; Leukemia ; immunology ; metabolism ; Leukocytes, Mononuclear ; metabolism ; Membrane Proteins ; metabolism ; Nod2 Signaling Adaptor Protein ; metabolism

IL-23 and IL-12 are structurally similar and critical for the generation of efficient cellular immune responses. Toxoplasma gondii induces a strong cell-mediated immune response. However, little is known about IL-23 secretion profiles in T. gondii-infected immune cells in connection with IL-12. We compared the patterns of IL-23 and IL-12 production by THP-1 human monocytic cells in response to stimulation with live or heat-killed T. gondii tachyzoites, or with equivalent quantities of either T. gondii excretory/secretory proteins (ESP) or soluble tachyzoite antigen (STAg). IL-23 and IL-12 were significantly increased from 6 hr after stimulation with T. gondii antigens, and their secretions were increased with parasite dose-dependent manner. IL-23 concentrations were significantly higher than those of IL-12 at the same multiplicity of infection. IL-23 secretion induced by live parasites was significantly higher than that by heat-killed parasites, ESP, or STAg, whereas IL-12 secretion by live parasite was similar to those of ESP or STAg. However, the lowest levels of both cytokines were at stimulation with heat-killed parasites. These data indicate that IL-23 secretion patterns by stimulation with various kinds of T. gondii antigens at THP-1 monocytic cells are similar to those of IL-12, even though the levels of IL-23 induction were significantly higher than those of IL-12. The detailed kinetics induced by each T. gondii antigen were different from each other.
Antigens, Protozoan/*immunology ; Cell Line ; Humans ; Interleukin-12/*secretion ; Interleukin-23/*secretion ; Monocytes/*immunology/*parasitology ; Time Factors ; Toxoplasma/*immunology

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

OBJECTIVETo investigate immunomodulatory effect of Astragalus polysaccharides (APS) on IL-12-secreting dendritic cell (DC) subset CD11c(high)CD45RB(low) DC.

METHODSSpleen CD11c(high)CD45RB(low) DC and CD4(+)T lymphocytes in BALB/c mice were purified by magnetic beads sorting, and were treated with 0 (as control), 50, 100, 200 µg/mL APS. Immunofluorescence staining and flow cytometry were used to determine expressions of CD11c(high)CD45RB(low) DC surface molecules, including CD40, CD80, CD86, I-A/E, and Toll-like receptor (TLR) 4. IL-12 level in CD11c(high)CD45RB(low) DC culture supernatant was determined by ELISA. The CD4(+) T lymphocytes were divided into: normal control group, non-stimulation group (CD4(+)T lymphocytes cocultured with APS-unstimulated CD11c(high)CD45RB(low) DC), high-dose APS stimulation group (CD4(+)T lymphocytes cocultured with 200 µg/mL APS-stimulated CD11c(high)CD45RB(low) DC), high-dose APS stimulation+antibody 1 group (CD4(+)T lymphocytes cocultured with 200 µg/mL APS-stimulated CD11c(high)CD45RB(low) DC and IL-12 antibody), high-dose APS stimulation+ antibody 2 group (CD4(+)T lymphocytes cocultured with 200 µg/mL APS-stimulated CD11c(high)CD45RB(low) DC and IL-12 antibody isotype). Proliferation ability of CD4(+) T lymphocytes was determined with MTT method. IL-4 level as well as IFN-γ level in CD4(+)T lymphocyte culture supernatant was determined by flow cytometry. Data were processed with one-way analysis of variance.

RESULTSCompared with those in control, the expressions of CD11c(high)CD45RB(low) DC surface molecules (except for CD86) on CD11c(high)CD45RB(low) DC surface, as well as IL-12-secreting level with dose-dependence were increased in cells stimulated with 50, 100, 200 µg/mL APS. Proliferation ability of CD4(+)T lymphocytes in high-dose APS stimulation group was higher as compared with that in non-stimulation group (F = 13.438, P < 0.05). IFN-γ level in high-dose APS stimulation group \[(2784 ± 137) pg/mL\] was higher than that in non-stimulation group \[(1952 ± 101) pg/mL, F = 12.177, P < 0.05\]. IL-4 level in high-dose APS stimulation group was (172 ± 20) pg/mL, which was lower than that in non-stimulation group \[(193 ± 19) pg/mL, F = 11.963, P < 0.05\]. Proliferation ability of CD4(+) T lymphocytes, IFN-γ level, and IL-4 level in high-dose APS stimulation + antibody 1 group were all ameliorated when compared with those in non-stimulation group; while levels of the 3 indexes in high-dose APS stimulation + antibody 2 group were similar to those in high-dose APS stimulation group.

CONCLUSIONSAPS can activate IL-12-producing CD11c(high)CD45RB(low) DC, and further induce the activation of immune function of T lymphocyte with shifting of Th2 to Th1 in vitro. APS can enhance the immune response via promoting the phenotypic and functional maturation of CD11c(high)CD45RB(low) DC.

Animals ; Astragalus Plant ; chemistry ; Cell Differentiation ; Cells, Cultured ; Dendritic Cells ; drug effects ; immunology ; secretion ; Interleukin-12 ; metabolism ; Mice ; Mice, Inbred BALB C ; Polysaccharides ; pharmacology ; Th1 Cells ; immunology

OBJECTIVETo prepare a vaccine of IL-12-anchored exosomes derived from renal cancer cells and to evaluate its antitumor effect in vitro.

METHODSA mammalian co-expression plasmid of glycolipid-anchor-IL-12 (GPI-IL-12) was constructed by subcloning IL-12A chain gene (P35 subunit) and a fusion gene containing GPI-anchor signal sequence and IL-12B chain gene (P40 subunit) in pBudCE4.1. Confocal laser scanning microscopy and flow cytometry were used to analyze the expression of the fusion proteins. Transmission electron microscopy and Western blot were used to identify the morphology and characteristic molecules of exosomes separated by ultrafiltration and sucrose gradient centrifugation. The function of IL-12-anchored exosomes was determined by IFN-gamma release assay.

RESULTSMammalian co-expression plasmids were successfully constructed. Confocal laser scanning microscopy and flow cytometric analysis of the RC-2-GPI-IL-12 transfectants showed the expression of IL-12 on the cell surface. Exosomes were purified by ultrafiltration and sucrose gradient centrifugation, which were 30-80 nm in diameter, typically saucer-shaped, and expressing HSP70, ICAM-1, G250 and GPI-IL-12. (80.0 +/- 9.6) pg/ml of IL-12 was detected in 10 microg/ml exosomes and it significantly induced the release of IFN-gamma. Stimulation with EXO-IL-12 could efficiently induce antigen-specific cytotoxic T lymphocytes (CTL), resulting in more significant cytotoxic effects in vitro.

CONCLUSIONA vaccine of exosomes-GPI-IL-12 can be obtained from the culture supernatant of renal cancer cells modified to express anchored IL-12. This vaccine expressing IL-12 and tumor associated antigen G250 may become a new strategy for the treatment of renal cancer.

Antigens, Neoplasm ; metabolism ; Cancer Vaccines ; immunology ; Carbonic Anhydrase IX ; Carbonic Anhydrases ; metabolism ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Exosomes ; genetics ; metabolism ; Glycosylphosphatidylinositols ; genetics ; metabolism ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Interferon-gamma ; secretion ; Interleukin-12 ; genetics ; metabolism ; Kidney Neoplasms ; metabolism ; pathology ; Plasmids ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection

This study was purposed to explore the changes in biological functions of human peripheral blood derived NK Cells after ex vivo expansion with different combinations of interleukin IL2 and/or IL12, IL15. According to different combination of cytokines, cultured NK cells were divided into 4 groups: group IL2, group IL2 + IL12, group IL2 + IL15 and group IL2 + IL15 + IL12. The group in which NK cells were cultured without cytokines was used as control. The cytotoxicity of cultured NK cells to target K562 cells was determined by using cell counting kit-8; the level of IFN-gamma in supernatants of NK cell culture was detected by ELISA; the perforin and granzyme B mRNA expressions were assayed by competitive quantitative RT-PCR. The results showed that the cytotoxicity of expanded NK cells in groups cultured with cytokines at different E:T ratio was significantly higher than that in group without cytokines (p < 0.01), although the cytotoxicity of NK cells in IL2 + IL15 + IL12 group seem to be slightly higher than that in IL2 + IL15 group, but there was no statistic difference (p > 0.05). The IFN-gamma levels in the supernatants of NK cell culture in the presence of cytokines significantly increased, and the IFN-gamma levels in IL2 + IL15 + IL12 group and IL2 + IL12 group were significantly higher than that in others (p < 0.01). The expressions of perforin and granzyme B mRNA of expanded NK cells in groups cultured with cytokines was significantly higher than that in control group (p < 0.01), and was consistent with cytotoxicity of NK cells. It is concluded that there are differences in the functions of NK cells cultured with different cytokines. IL2 and IL15 have synergistic effect on strengthening cytotoxicity of NK cells and promoting cell expansion. However, the main function of IL12 promotes NK cells to secrete IFN-gamma, which plays a role in immunoregulation.
Humans ; Interferon-gamma ; secretion ; Interleukin-12 ; administration & dosage ; pharmacology ; Interleukin-15 ; administration & dosage ; pharmacology ; Interleukin-2 ; administration & dosage ; pharmacology ; K562 Cells ; Killer Cells, Natural ; drug effects ; immunology

OBJECTIVETo investigate the antitumor effects of cytotoxic T lymphocytes (CTLs) induced by autologous dendritic cells that were inspired by autologous tumor lysates (ATLs-mDCs).

METHODSPrimary gastric cancer cells prepared by short-term culture were used as targets. ATLs-mDCs were subjected to activate autologous T cells to generate CTLs. The immunological functions of DCs were evaluated by flow cytometry and by mixed leukocyte response (MLR) assay. The antitumor outcome of tumor antigen specific CTLs was tested by cytotoxicity assay. Concentrations of IL-12 in cultured DCs and INF-gamma in CTLs were measured by ELISA.

RESULTSThe expressions of MHC-II, CD80, CD83 and CD86 were significantly up-regulated in ATLs-mDCs, moreover, the ATLs-mDCs obtained the capability of stimulating the proliferation of autologous T cells with high efficiency. The secretion of IL-12 in ATLs-mDCs was significantly higher than that in pure mature DCs (t = 15.47, P < 0.01) and in immature DCs (t = 28.44, P < 0.01). The secretion of INF-gamma in CTLs activated by ATLs-mDCs was significantly higher than that in CTLs by pure mature DCs (t = 4.84, P < 0.05) and in CTLs by immature DCs (t = 13.74, P < 0. 01). The antigen specific cytotoxicity of CTLs induced by ATLs-mDCs was significantly higher against autologous tumor cells [(84 +/- 11)%] than that against two allogeneic tumor cell lines [(19 +/- 7)% and (19 +/- 11)%; t = 54.18 and 56.46, P < 0.01, respectively].

CONCLUSIONSATLs-mDCs might mediate the antigen specific CTLs against autologous gastric cancer cells ex vivo with high efficiency.

Cytotoxicity, Immunologic ; Dendritic Cells ; immunology ; Humans ; Immunotherapy, Adoptive ; methods ; In Vitro Techniques ; Interferon-gamma ; secretion ; Interleukin-12 ; secretion ; Stomach Neoplasms ; therapy ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo study the effect of bifidobacterium genomic DNA on umbilical cord blood mononuclear cell (CBMC), and investigate the immunoregulation of bifidobacterium DNA and explore possible mechanisms by which bifidobacterium acts against allergic reaction.

METHODSBifidobacterium genomic DNA (bDNA) and human DNA (hDNA) were extracted with phenol/chloroform/isoamyl alcohol and stored at -20 degrees C for later use. Parts of bDNA were completely digested with DNaseI (d-bDNA) at 37 degrees C. CBMCs were isolated with Ficoll from umbilical cord blood and incubated at 37 degrees C in a 5% CO2 humidified incubator. These cells were divided into four groups, control group: without any stimulant; bDNA group: stimulated with 25 microg/ml bDNA; d-bDNA group: stimulated with 25 microg/ml d-bDNA; hDNA group: stimulated with 25 microg/ml hDNA. The cells were stimulated with different stimulants in vitro, at the end of incubation culture supernatant and cells were collected. IL-12 and IL-10 levels in the culture supernatant were measured by enzyme linked immuno sorbent assay (ELISA); cells secreting IL-4 and IFN-gamma were counted by enzyme linked immunospot (ELISPOT) assay; and total RNA was isolated from the cells to assay T-bet and GATA3 mRNA expression levels by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSSix hours after stimulation there was no significant difference in IL-12 level in supernatant among the four groups; 12 hours after stimulation, IL-12 level in supernatant of bDNA treated group was significantly higher than that of each of the other groups, so were the results obtained at 24 hours and 48 hours after stimulation (P < 0.05). No significant difference could be detected in IL-12 level in supernatant among the other 3 groups. On the other hand, 6 hours after stimulation there was no significant difference in IL-10 level in supernatant among the four groups. But 12 and 24 hours after stimulation IL-10 level in supernatant of bDNA treated group was lower than that of each of the other groups, but the difference was not statistically significant. The count of IFN-gamma secreting cells of bDNA treated group was higher than that of the other groups, while IL-4 secteting cells of bDNA treated group were lower than that of the other groups. After bDNA stimulation, nuclear factor T-box expressed in T cells (T-bet) mRNA expression was conspicuously enhanced as compared to the other three groups (P < 0.05). GATA3 mRNA transcription in CBMC had no significant change after bDNA stimulation.

CONCLUSIONbDNA could promote secretion of Th1 type cytokine IL-12, while Th2 type cytokine IL-10 level of cell supernatant was decreased. bDNA could stimulate secretion of IFN-gamma by CBMC and inhibit secretion of IL-4. T-bet mRNA expression was highly enhanced after bDNA stimulation. bDNA could upregulate Th1 type response, which may be one of important mechanisms by which bifidobacterium inhibit allergic response.

Bifidobacterium ; cytology ; genetics ; Cell Culture Techniques ; DNA, Bacterial ; biosynthesis ; metabolism ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Fetal Blood ; cytology ; immunology ; GATA3 Transcription Factor ; genetics ; Humans ; Infant, Newborn ; Interferon-gamma ; immunology ; secretion ; Interleukin-10 ; immunology ; secretion ; Interleukin-12 ; immunology ; secretion ; Interleukin-4 ; immunology ; secretion ; Leukocytes, Mononuclear ; immunology ; secretion ; RNA, Messenger ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; T-Box Domain Proteins ; genetics ; Th1 Cells ; drug effects ; immunology ; secretion

OBJECTIVETo investigate the effects of dendritic cells (DCs) transfected with survivin gene, and to observe the effective and specific anti-tumor immunological effect induced by modified DC in vitro.

METHODSSurvivin gene was transfected to DCs with liposomes. Survivin expression could be detected both in DCs cells and in cell culture with method of Western blot. Cytokines as well as cellular surface molecule such as IL-12, TNF-alpha, CD1 alpha, CD83, MHCII, CD80 and CD86 were detected. The competence of inducing human specific cytotoxic T lymphocyte (CTLs) was also detected with MTT.

RESULTSSurvivin expression could be detected both in DCs which were transfected with survivin cDNA and in cell culture superior. The IL-12 and TNF-alpha level was (265.2 +/- 32.7), (437.1 +/- 83.5) pg/ml, and much higher in transgened DC cells than blank DC cells (P < 0.05). CD1 alpha, CD83, MHCII, CD80 and CD86 was high expressed in survivin-DC cells, however, it was low expressed in blank DC cells. The lyse rate to gastric cancer cell, colon cancer cell and bile duct cancer cell was 65%, 77%, and 85% respectively, and these were much higher than those of blank DC cells.

CONCLUSIONSDCs transfected with survivin gene could induce specific cytotoxic T lymphocytes and strikingly raised DC cell's antigen present function, and have specific CTL killing activity.

Antigens, CD ; metabolism ; Dendritic Cells ; immunology ; Gastrointestinal Neoplasms ; therapy ; Humans ; Immunotherapy, Active ; In Vitro Techniques ; Inhibitor of Apoptosis Proteins ; Interleukin-12 ; secretion ; Microtubule-Associated Proteins ; genetics ; Neoplasm Proteins ; genetics ; Transfection ; Tumor Necrosis Factor-alpha ; secretion

Cell Survival ; drug effects ; Cells, Cultured ; Dendritic Cells ; drug effects ; immunology ; physiology ; Humans ; Immunophenotyping ; Interleukin-10 ; pharmacology ; Interleukin-12 ; genetics ; secretion ; Interleukin-12 Subunit p35 ; Interleukin-12 Subunit p40 ; Protein Subunits ; genetics ; secretion ; RNA, Messenger ; analysis