Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells

Mesenchymal stem cells (MSCs), in addition to their multilineage differentiation, have a direct immunosuppressive effect on T-cell proliferation in vitro. However, it is unclear whether they also modulate the immune system by acting on the very first step. In this investigation, we addressed the effects of human MSCs on the differentiation, maturation, and function of dendritic cells (DCs) derived from CD14+ monocytes in vitro. Upon induction with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4), MSC coculture could strongly inhibit the initial differentiation of monocytes to DCs, but this effect is reversible. In particular, such suppression could be recapitulated with no intercellular contact at a higher MSC/monocyte ratio (1:10). Furthermore, mature DCs treated with MSCs were significantly reduced in the expression of CD83, suggesting their skew to immature status. Meanwhile, decreased expression of presentation molecules (HLA-DR and CD1a) and costimulatory molecules (CD80 and CD86) and down-regulated IL-12 secretion were also observed. In consistence, the allostimulatory ability of MSC-treated mature DCs on allogeneic T cells was impaired. In conclusion, our data suggested for the first time that human MSCs could suppress monocyte differentiation into DCs, the most potent antigen-presenting cells (APCs), thus indicating the versatile regulation of MSCs on the ultimate specific immune response.     

Generation of CD1+RelB+ dendritic cells and tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated giant cells from human monocytes

We previously showed that granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) stimulate the differentiation of human monocytes into two phenotypically distinct types of macrophages. However, in vivo, not only CSF but also many other cytokines are produced under various conditions. Those cytokines may modulate the differentiation of monocytes by CSFs. In the present study, we showed that CD14+ adherent human monocytes can differentiate into CD1+relB+ dendritic cells (DC) by the combination of GM-CSF plus interleukin-4 (IL-4) and that they differentiate into tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like multinucleated giant cells (MGC) by the combination of M-CSF plus IL-4. However, the monocyte-derived DC were not terminally differentiated cells; they could still convert to macrophages in response to M-CSF. Tumor necrosis factor-alpha (TNF-alpha) stimulated the terminal differentiation of the DC by downregulating the expression of the M-CSF receptor, cfms mRNA, and aborting the potential to convert to macrophages. In contrast to IL-4, interferon-gamma (IFN-gamma) had no demonstrable effect on the differentiation of monocytes. Rather, IFN- gamma antagonized the effect of IL-4 and suppressed the DC and MGC formation induced by GM-CSF + IL-4 and M-CSF + IL-4, respectively. Taken together, these results provide a new aspect to our knowledge of monocyte differentiation and provide evidence that human monocytes are flexible in their differentiation potential and are precursors not only of macrophages but also of CD1+relB+DC and TRAP-positive MGC. Such a diverse pathway of monocyte differentiation may constitute one of the basic mechanisms of immune regulation.     

Prolongation of liver allograft survival by dendritic cells modified with NF-κB decoy oligodeoxynucleotides

AIM: To induce the tolerance of rat liver allograft by dendritic cells (DCs) modified with NF-κB decoy oligodeoxynucleotides (ODNs).METHODS: Bone marrow (BM)-derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF+IL-4 to obtain immature DCs or mature DCs. GM-CSF+IL-4-propagated DCs were treated with double-strand NF-κB decoy ODNs containing two NF-κB binding sites or scrambled ODNs to ascertain whether NF-κB decoy ODNs might prevent DC maturation. GM-CSF-propagated DCs, GM-CSF+NF-κB decoy ODNs or scrambled ODNs-propagated DCs were treated with LPS for 18 h to determine whether NF-κB decoy ODNs could prevent LPS-induced IL-12 production in DCs. NF-κB binding activities, costimulatory molecule (CD40, CD80, CD86) surface expression, IL-12 protein expression and allostimulatory capacity of DCs were measured with electrophoretic mobility shift assay (ENSA),flow cytometry, Western blotting, and mixed lymphocyte reaction (MLR), respectively. GM-CSF-propagated DCs, GM-CSF+IL-4 -propagated DCs, and GM-CSF+NF-κB decoy ODNs or scrambled ODNs-propagated DCs were injected intravenously into recipient LEW rats 7 d prior to liver transplantation and immediately after liver transplantation.Histological grading of liver graft rejection was determined 7 d after liver transplantation. Expression of IL-2, IL-4 and IFN-γ, mRNA in liver graft and in recipient spleen was analyzed by semiquantitative RT-PCR. Apoptosis of liver allograft-infiltrating cells was measured with TUNEL staining.RESULTS: GM-CSF-propagated DCs, GM-CSF+NF-κB decoy ODNs-propagated DCs and GM-CSF+ scrambled ODNs-propagated DCs exhibited features of immature DCs, with similar low level of costimulatory molecule(CD40, CD80,CD86) surface expression, absence of NF-κB activation,and few allocostimulatory activities. GM-CSF+IL-4-propagated DCs displayed features of mature DCs, with high levels of costimulatory molecule (CD40, CD80, CD86) surface expression, marked NF-κB activation, and significant allocostimulatory activity. NF-κB decoy ODNs completely abrogated IL-4-induced DC maturation and allocostimulatory activity as well as LPS-induced NF-κB activation and IL-12 protein expression in DCs. GM-CSF+NF-κB decoy ODNs-propagated DCs promoted apoptosis of liver allograft-infiltrating cells within portal areas, and significantly decreased the expression of IL-2 and IFN-γ mRNA but markedly elevated IL-4 mRNA expression both in liverallograft and in recipient spleen, and consequently suppressed liver allograft rejection, and promoted liverallograft survival.CONCLUSION: NF-κB decoy ODNs-rnodified DCs canprolong liver allograft survival by promoting apoptosis of graft-infiltrating cells within portal areas as well as down-regulating IL-2 and IFN-γ mRNA and up-regulating IL-4 rnRNA expression both in liver graft and in recipient spleen.     

Phenotypic and Functional Comparison between Flask Adherent and Magnetic Activated Cell Sorted Monocytes Derived Dendritic Cells

Background: Generation of an effective dendritic cell (DC) based cancer vaccine depends on appropriate differentiation of monocytes in vitro. Objective: To compare the effects of monocyte separation methods, flask adherence (Flask-DC) and magnetic activated cell sorting (MACS-DC), on phenotypic and functional characteristics of resultant DCs. Methods: DCs from healthy volunteers were generated from plastic adherence and MACS isolated monocytes in the presence of GM-CSF and IL-4 as well as TNF-α and monocyte conditioned medium (MCM) in 7 day cultures. Mature DCs were then subjected to phenotypic analysis using anti-CD14, anti-CD83 and HLA-DR monoclonal antibodies. Functional and cytokine release assays were carried out using [3H] thymidine uptake test and commercially available ELISA kits for the determination of IL-12, IL-10, IFN-γ and IL-4, respectively. Results: We found that MACS-DCs were more homogenous and the yield and viability were fairly higher than Flask-DCs. MACS-DCs expressed higher levels of CD83 and HLA-DR as well as CD14 compared to the Flask-DCs. Induction of T cell proliferative responses were higher in Flask-DCs and also they elicited higher levels of IL-12: IL-10 and IFN-γ: IL-4 ratios in cytokine generation assays. Conclusion: MACS method was superior for mass production of viable homogenous and fully mature DCs but their cytokine profile had the potential to polarize the immune system toward Th2 type immune response.     

1,25-Dihydroxyvitamin D(3) inhibits dendritic cell differentiation and maturation in vitro

OBJECTIVE: Because of its potent immunosuppressive properties in vitro as well as in vivo, we studied the effect of 1,25-dihydroxyvitamin D(3) (calcitriol) on differentiation, maturation, and function of dendritic cells (DC). MATERIALS AND METHODS: Monocyte-derived DCs were generated with GM-CSF plus IL-4, and maturation was induced by a 2-day exposure to TNFalpha. DCs were derived from CD34(+) progenitors using SCF plus GM-CSF plus TNFalpha. For differentiation studies, cells were exposed to calcitriol at concentrations of 10(-)(9)- 10(-7) M at days 0, 6, and 8, respectively. The obtained cell populations were evaluated by morphology, phenotype, and function. RESULTS: When added at day 0, calcitriol blocked DC differentiation from monocytes and inhibited the generation of CD1a(+) cells from progenitor cells while increasing CD14(+) cells. Exposure of immature DCs to calcitriol at day 6 resulted in a loss of the DC-characteristic surface molecule CD1a, downregulation of the costimulatory molecules CD40 and CD80, and MHC class II expression, whereas the monocyte/macrophage marker CD14 was clearly reinduced. In addition, calcitriol hindered TNFalpha-induced DC maturation, which is usually accompanied with induction of CD83 expression and upregulation of costimulatory molecules. In contrast, the mature CD83(+) DCs remained CD1a(+)CD14(-) when exposed to calcitriol. The capacity of cytokine-treated cells to stimulate allogeneic and autologous T cells and to take up soluble antigen was inhibited by calcitriol. CONCLUSION: The potent suppression of DC differentiation, the reversal of DC phenotype, and function in immature DCs, as well as the inhibition of DC maturation by calcitriol, may explain some of its immunosuppressive properties.     

Functional changes of dendritic cells derived from allogeneic partial liver graft undergoing acute rejection in rats

AIM: To investigate functional change of dendritic cells (DCs) derived from allogeneic partial liver graft undergoing acute rejection in rats. METHODS: Allogeneic (SD rat to LEW rat) whole and 50 % partial liver transplantation were performed. DCs from liver grafts 0 hr and 4 days after transplantation were isolated and propagated in the presence of GM-CSF in vitro. Morphological characteristics of DCs propagated for 4 days and 10 days were observed by electron microscopy. Phenotypical features of DCs propagated for 10 days were analyzed by flow cytometry. Expression of IL-12 protein and IL-12 receptor mRNA in DCs propagated for 10 days was also measured by Western blotting and semiquantitative RT-PCR, respectively. Histological grading of rejection were determined. RESULTS: Allogeneic whole liver grafts showed no features of rejection at day 4 after transplantation. In contrast, allogeneic partial liver grafts demonstrated moderate to severe rejection at day 4 after transplantation. DCs derived from allogeneic partial liver graft 4 days after transplantation exhibited typical morphological characteristics of DC after 4 days' culture in the presence of GM-CSF. DCs from allogeneic whole liver graft 0 hr and 4 days after transplantation did not exhibit typical morphological characteristics of DC until after 10 days' culture in the presence of GM-CSF. After 10 days' propagation in vitro, DCs derived from allogeneic whole liver graft exhibited features of immature DC, with absence of CD40, CD80 and CD86 surface expression, and low levels of IL-12 proteins (IL-12 p35 and IL-12 p40) and IL-12 receptor (IL-12Rbeta(1) and IL-12Rbeta(2)) mRNA, whereas DCs from allogeneic partial liver graft 4 days after transplantation displayed features of mature DC, with high levels of CD40, CD80 and CD86 surface expression, and as a consequence, higher expression of IL-12 proteins (IL-12 p35 and IL-12 p40) and IL-12 receptors (IL-12Rbeta1 and IL-12Rbeta2) mRNA than those of DCs both from partial liver graft 0 hr and whole liver graft 4 days after transplantation (P<0.001) was observed. CONCLUSION: DCs derived from allogeneic partial liver graft undergoing acute rejection display features of mature DC.     

Prolongation of liver allograft survival by dendritic cells modified with NF-κB decoy oligodeoxynucleotides

AIM: To induce the tolerance of rat liver allograft by dendritic cells (DCs) modified with NF-κB decoy oligodeoxynucleotides (ODNs).METHODS: Bone marrow (BM)-derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF+IL-4to obtain immature DCs or mature DCs. GM-CSF+IL-4-propagated DCs were treated with double-strand NF-κB decoy ODNs containing two NF-κB binding sites or scrambled ODNs to ascertain whether NF-κB decoy ODNs might prevent DC maturation. GM-CSF-propagated DCs, GMCSF+NF-κB decoy ODNs or scrambled ODNs-propagated DCs were treated with LPS for 18 h to determine whether NF-κB decoy ODNs could prevent LPS-induced IL-12production in DCs. NF-κB binding activities, costimulatory molecule (CD40, CD80, CD86) surface expression, IL-12protein expression and allostimulatory capacity of DCs were measured with electrophoretic mobility shift assay (EMSA),flow cytometry, Western blotting, and mixed lymphocyte reaction (MLR), respectively. GM-CSF-propagated DCs, GMCSF+IL-4 -propagated DCs, and GM-CSF+NF-κB decoy ODNs or scrambled ODNs-propagated DCs were injected intravenously into recipient LEW rats 7 d prior to liver transplantation and immediately after liver transplantation.Histological grading of liver graft rejection was determined 7 d after liver transplantation. Expression of IL-2, IL-4 and IFN-γ mRNA in liver graft and in recipient spleen was analyzed by semiquantitative RT-PCR. Apoptosis of liver allograft-infiltrating cells was measured with TUNEL staining.RESULTS: GM-CSF-propagated DCs, GM-CSF+NF-κB decoy ODNs-propagated DCs and GM-CSF+ scrambled ODNspropagated DCs exhibited features of immature DCs, with similar low level of costimulatory molecule(CD40, CD80,CD86) surface expression, absence of NF-κB activation,and few allocostimulatory activities. GM-CSF+IL-4-propagated DCs displayed features of mature DCs, with high levels of costimulatory molecule (CD40, CD80, CD86) surface expression, marked NF-κB activation, and significant allocostimulatory activity. NF-κB decoy ODNs completely abrogated IL-4-induced DC maturation and allocostimulatory activity as well as LPS-induced NF-κB activation and IL-12protein expression in DCs. GM-CSF+NF-κB decoy ODNspropagated DCs promoted apoptosis of liver allograftinfiltrating cells within portal areas, and significantly decreased the expression of IL-2 and IFN-γ mRNA but markedly elevated IL-4 mRNA expression both in liver allograft and in recipient spleen, and consequently suppressed liver allograft rejection, and promoted liver allograft survival.CONCLUSION: NF-κB decoy ODNs-modified DCs can prolong liver allograft survival by promoting apoptosis of graft-infiltrating cells within portal areas as well as downregulating IL-2 and IFN-γ mRNA and up-regulating IL-4 mRNA expression both in liver graft and in recipient spleen.     

Efficient Ex Vivo Generation of Human Dendritic Cells from Mobilized CD34p+ Peripheral Blood Progenitors

We tried to efficiently generate human dendritic cells (DCs) from CD34+ peripheral blood hematopoietic progenitor cells mobilized by high-dose chemotherapy and subsequent administration of granulocyte colony-stimulating factor, using a liquid suspension culture system. Among various combinations, the combination of c-kit ligand, flt-3 ligand, c-mpl ligand (TPO), and interleukin (IL)-4 most potently generated the number of CD1a+CD14- DCs in cultures containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha). The delayed addition of IL-4 on day 6 of culture gave rise to an additional increase in the yield of CD1a+CD14-DCs that were characterized by the expression of HLA-ABC, HLA-DR, CD80, CD86, and CD83. The majority of the sorted CD1a-CD14+ cells derived from 6-day culture of CD34+ cells gave rise to CD1a+CD14- DCs and CD1a-CD14+ macrophages on day 12 of culture in the presence and absence of IL-4, respectively. These findings suggest that IL-4 promotes the differentiation of CD1a- CD14+ cells derived from mobilized CD34+ peripheral blood hematopoietic progenitors to CD1a+ CD14- DCs. The majority of these DCs expressed CD68 but not the Langerhans-associated granule antigen, a finding that suggests they emerge through the monocyte differentiation pathway. The addition of TPO and IL-4 to cultures did not affect the potential of DCs to stimulate the primary allogeneic T-cell response. These findings demonstrated that the combination of c-kit ligand plus flt-3 ligand plus TPO with GM-CSF plus TNF-alpha, followed by IL-4, is useful for ex vivo generation of human DCs from mobilized CD34+ peripheral blood progenitors.     

Regulatory dendritic cells program generation of interleukin-4-producing alternative memory CD4 T cells with suppressive activity

The heterogeneity and mechanisms for the generation of CD4 memory T (CD4 Tm) cells remain elusive. Distinct subsets of dendritic cells (DCs) have been found to regulate a distinct T-helper (Th)-cell subset differentiation by influencing cytokine cues around CD4 T cells; however, whether and how the regulatory DC subset can regulate Tm-cell differentiation remains unknown. Further, there is no ideal in vitro experimental system with which to mimic the 3 phases of the CD4 T-cell immune response (expansion, contraction, memory generation) and/or to culture CD4 Tm cells for more than a month. By analyzing CD4 T cells programmed by long-term coculture with regulatory DCs, we identified a population of long-lived CD4 T cells with a CD44(hi)CD62L(-)CCR7(-) effector memory phenotype and rapid, preferential secretion of the Th2 cytokines interleukin-4 (IL-4), IL-5, IL-10, and IL-13 after antigenic stimulation. These regulatory DC-programmed Tm cells suppress CD4 T-cell activation and proliferation in vitro via IL-10 and inhibit the delayed-type hypersensitivity response once infused in vivo. We also identify their natural counterpart, which is up-regulated by regulatory DC transfusion and negatively regulates the recall response in vivo. Different from interferon-γ-producing conventional Tm cells, these IL-4-producing CD4 Tm cells act as alternative Tm cells with a regulatory function, suggesting a new way of negative immune regulation by memory T cells.     

Monocyte-derived dendritic cells from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine profile.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is characterized by an insufficient immune response, possibly owing to impaired function of antigen-presenting cells such as myeloid dendritic cells (DCs). Therapeutic vaccination with in vitro generated DCs may enhance the immune response. Subsets of DCs can originate from monocytes, but the presence of HCV in monocytes that develop into DCs in vitro may impair DC function. Therefore, we studied the presence of HCV RNA in monocytes and monocyte-derived DCs from chronic HCV patients. METHODS: Monocytes were cultured with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) for 6 days, and then with GM-CSF, IL-4, tumour necrosis factor-alpha (TNF-alpha), prostaglandin E2, IL-1beta and IL-6 for 2 days to generate mature DCs. HCV RNA was assessed by polymerase chain reaction. Surface molecules were assessed by flow cytometry. Cytokine production was assessed by cytokine bead array. RESULTS: HCV RNA was present in monocytes in 11 of 13 patients, but undetectable in mature DCs in 13 of 13 patients. The morphology of patient DCs was comparable with DCs from healthy controls, but the percentage of cells expressing surface molecules CD83 (P=0.001), CD86 (P=0.023) and human leucocyte antigen-DR (P=0.028) was lower in HCV patients. Compared with control DCs, patient DCs produced enhanced levels of IL-10 (P=0.0079) and IL-8 (P=0.0079), and lower levels of TNF-alpha (P=0.032), IL-6 (P=NS) and IL-1beta (P=0.0079). Patient and control DCs did not produce IL-12. CONCLUSIONS: Monocyte-derived DCs from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine profile.     

Arrest of human dendritic cells at the CD34-/CD4+/HLA-DR+ stage in the bone marrow of NOD/SCID-human chimeric mice

Human dendritic cell (DC) precursors were engrafted and maintained in NOD/SCID- human chimeric mice (NOD/SCID-hu mice) implanted with human cord blood mononuclear cells, although no mature human DCs were detected in lymphoid organs of the mice. Two months after implantation, bone marrow (BM) cells of NOD/SCID-hu mice formed colonies showing DC morphology and expressing CD1a in methylcellulose culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha). The CD34-/CD4+/HLA-DR+ cell fraction in NOD/SCID-hu mouse BM generated CD1a(+) cells that were highly stimulatory in mixed leukocyte reactions in culture with GM-CSF and TNF-alpha. These results suggest a strong potential for NOD/SCID-hu BM to generate human DCs, although DC differentiation may be blocked at the CD34-/CD4+/HLA-DR+ stage. (Blood. 2001;97:3655-3657)     

Adenosine receptors in regulation of dendritic cell differentiation and function

Differentiation of functional dendritic cells (DCs) critically depends on the microenvironment. DCs differentiate in hypoxic tumor sites and inflamed or damaged tissue. Because local concentrations of adenosine reach high physiologically relevant levels in these conditions, we assessed the expression of adenosine receptors and the effect of their activation on differentiation of human monocytes and mouse peritoneal macrophages and hematopoietic progenitor cells (HPCs) into myeloid DCs. Stimulation of adenosine receptors skews DC differentiation toward a distinct cell population characterized by expression of both DC and monocyte/macrophage cell surface markers. Pharmacologic analysis and experiments with cells from A(2B) adenosine receptor knockout mice identified A(2B) receptor as the mediator of adenosine effects on DCs. Unlike normal myeloid DCs, adenosine-differentiated DCs have impaired allostimulatory activity and express high levels of angiogenic, pro-inflammatory, immune suppressor, and tolerogenic factors, including VEGF, IL-8, IL-6, IL-10, COX-2, TGF-beta, and IDO. They promoted tumor growth if injected into tumors implanted in mice. Using adenosine desaminase knockout animals, we showed that DCs with proangiogenic phenotype are highly abundant under conditions associated with elevated levels of extracellular adenosine in vivo. Adenosine signaling through A(2B) receptor is an important factor of aberrant DC differentiation and generation of tolerogenic, angiogenic, and proinflammatory cells.     

Expression of CCR-7, MIP-3beta, and Th-1 chemokines in type I IFN-induced monocyte-derived dendritic cells: importance for the rapid acquisition of potent migratory and functional activities.

The migration capability of dendritic cells (DCs) is regulated by their response to factors, namely chemokines, that characterize maturation stage and shape their functional activities. This study examines the morphology, expression of chemokines/chemokine receptors, and migration properties of DCs generated after treatment of monocytes with type I interferon (IFN) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (IFN-DCs). IFN-DCs showed phenotypical and morphologic features undetectable in DCs generated in the presence of interleukin 4 (IL-4) and GM-CSF, such as expression of CD83 and CD25 and the presence of CD44+, highly polarized, thin, and long dendrites. IFN-DCs markedly migrated in response to beta-chemokines (especially MIP-1beta) and expressed the Th-1 chemokine IP-10. Notably, IFN-DCs showed an up-regulation of CCR7 as well as of its natural ligand MIP-3beta, characteristics typical of mature DCs. Of interest, IFN-DCs exhibited a marked chemotactic response to MIP-3beta in vitro and strong migratory behavior in severe combined immunodeficient (SCID) mice. In SCID mice reconstituted with human peripheral blood leukocytes, IFN-DCs induced a potent primary human antibody response and IFN-gamma production, indicative of a Th-1 immune response. These results define the highly specialized maturation state of IFN-DCs and point out the existence of a "natural alliance" between type I IFN and monocyte/DC development, instrumental for ensuring an efficient connection between innate and adaptive immunity.     

Phenotypic analysis of circulating and intrahepatic dendritic cell subsets in patients with chronic liver diseases

BACKGROUND/AIMS: Dendritic cells (DCs) are the most potent professional antigen-presenting cells. Although two subsets of circulating DCs, lineage(-)CD11c(+)CD4(low) (CD11c(+)DCs) and lineage (-)CD11c(-)CD4(+)CD123(+) (CD123(+)DCs) are identified in humans, the role of each DC subset in the immunopathogenesis of liver diseases is unknown.METHODS: We examined the numbers and activation status of each DC subset in the circulation and in the inflamed livers in patients with chronic liver diseases by flow cytometry and immunohistochemistry.RESULTS: The numbers of circulating CD11c(+)DCs were inversely correlated with serum alanine aminotransferase (ALT) levels in patients with chronic viral hepatitis, and that the expression of costimulatory molecules on circulating CD11c(+)DCs in patients with chronic viral hepatitis was significantly up-regulated in patients with high serum levels of ALT. Both DCs are also identified in the livers by flow cytometry, and the expression of costimulatory molecule CD40 on those DCs was significantly higher in liver DCs than that in circulating DCs. Moreover, the ratios of CD11c(+)DCs/CD123(+)DCs were higher in liver DCs (mean+/-SD, 7.2+/-6.0) than those of circulating DCs (4.0+/-4.6). Immunohistochemically, CD11c(+) or CD123(+) cells and CD83(+) activated DCs were observed mostly in portal areas with mononuclear cell infiltration in various liver diseases. These overall data suggest that DCs, especially CD11c(+)DCs, could be associated with the necroinflammatory response in the liver of chronic viral liver diseases.CONCLUSIONS: DCs, especially CD11c(+)DCs, may be involved in the immunopathogenesis of chronic liver diseases.     

来氟米特对狼疮患者树突状细胞作用机制的初探

目的　探讨来氟米特(LEF)处理前后系统性红斑狼疮(SLE)患者树突状细胞(DC)表面标志及功能的改变,揭示LEF治疗SLE的作用机制,为开展“抑制性DCs”治疗SLE奠定实验基础。方法　(1)分离SLE患者外周血单核细胞,用细胞因子诱导DC成熟, LEF组再加入A7717262(来氟米特的活性代谢产物)培养。第9天收集DC细胞,流式细胞仪检测CD80、CD83、CD86和HLA DR的表达。(2)分别将A771726处理或不处理的第9天DC和T细胞进行培养, 72h后用MTT法检测DC刺激淋巴细胞增殖的能力,FACS检测T细胞亚群和ELISA检测培养上清中IL 10和IFNγ水平。结果A771726处理后虽DC形态无改变,但DC表达CD83、CD86和HLA DR百分数较对照组均明显降低(72 70±1 77vs 79 36±4 80, 63 50±14 06vs. 83 91±9 81, 80 44±12 56vs. 90 51±8 63,P值均<0 01)。A771726处理后的DC,其刺激T细胞增殖相应的吸光度值明显降低,混合培养的上清液中IL 10水平较无A771726处理的DC与T细胞的混合培养上清液明显降低,而IFNγ两者间无显著差异;但见CD 4 CD 25CTLA 4 T细胞百分比增高。结论　LEF在体外可抑制SLE患者外周血DC的成熟;未成熟DC能抑制T细胞增殖及T细胞向Th2 细胞转化,诱导CD 4 CD 25CTLA 4 T细胞产生,从而纠正SLE患者的部分免疫紊乱。     

Complement-induced regulatory T cells suppress T-cell responses but allow for dendritic-cell maturation

Concurrent activation of the T-cell receptor (TCR) and complement regulator CD46 on human CD4+ T lymphocytes induces Tr1-like regulatory T cells that suppress through IL-10 secretion bystander T-cell proliferation. Here we show that, despite their IL-10 production, CD46-induced T-regulatory T cells (Tregs) do not suppress the activation/maturation of dendritic cells (DCs). DC maturation by complement/CD46-induced Tregs is mediated through simultaneous secretion of GM-CSF and soluble CD40L, factors favoring DC differentiation and reversing inhibitory effects of IL-10. Thus, CD46-induced Tregs produce a distinct cytokine profile that inhibits T-cell responses but leaves DC activation unimpaired. Such "DC-sparing" Tregs could be desirable at host/environment interfaces such as the gastrointestinal tract where their specific cytokine profile provides a mechanism that ensures unresponsiveness to commensal bacteria while maintaining reactivity to invading pathogens.     

Dendritic cells generated in the presence of granulocyte-macrophage colony-stimulating factor and IFN-alpha are potent inducers of HIV-specific CD8 T cells

OBJECTIVE: To investigate the ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-alpha to induce the differentiation of peripheral monocytes into dendritic cells (DC) and their ability to trigger an HIV-specific CD8 T-cell response. METHODS: Monocytes isolated from both seronegative controls and HIV-infected individuals were differentiated into DC using GM-CSF with either IL-4 or IFN-alpha for 7 days. We assessed the phenotypic characteristics and IL-12 production by flow cytometry. The ability of DC to trigger CD8 T-cell responses was assessed by means of ELISpot and cytotoxicity assays. In addition, HIV-1-RNA levels were measured in culture supernatants. RESULTS: Compared with control DC generated in the presence of GM-CSF and IL-4, DC generated in the presence of GM-CSF and IFN-alpha expressed higher levels of MHC class I molecules and produced similar or higher levels of IL-12 after CD40 ligation or Staphyloccus aureus Cowan stimulation. GM-CSF/IFN-alpha DC expressed low levels of CD4, CXCR4 and DC-SIGN and did not produce detectable virus during the differentiation period. Pulsed GM-CSF/IFN-alpha DC were found to prime CD8 T cells from HIV-negative controls to exert cytotoxic activity against target cells expressing HIV antigens. HIV peptide-pulsed GM-CSF/IFN-alpha DC promote specific IFN-gamma production by autologous CD8 T cells from HIV-seronegative donors. Furthermore, GM-CSF/IFN-alpha DC from HIV-seropositive patients efficiently present HIV peptides to autologous CD8 T lymphocytes. CONCLUSION: GM-CSF and IFN-alpha allow the generation of DC with high CD8 T-cell stimulating abilities. Therefore, this strategy may represent a novel approach to therapeutic vaccination in HIV disease.