Human Blood Dendritic Cells Research Articles

Bortezomib significantly impairs the immunostimulatory capacity of human myeloid blood dendritic cells

Bortezomib is a potent drug for the treatment of multiple myeloma. Its anti-tumor activity is mediated by proteasome inhibition leading to decreased cell proliferation and induction of apoptosis. However, an unimpaired proteasomal function plays a crucial role for the induction of anti-tumor immunity by dendritic cells (DCs), which are currently used for therapeutic vaccination against various tumors including myeloma. In the present study, we investigated the impact of bortezomib on the immunostimulatory capacity of 6-sulfo LacNAc (slan) DCs, which represent a major subset of human blood DCs. We demonstrated that this proteasome inhibitor efficiently impairs the spontaneous in vitro maturation of slanDCs and the release of tumor necrosis factor (TNF)-alpha as well as interleukin (IL)-12 upon lipopolysaccharide (LPS) stimulation. Functional data revealed that bortezomib profoundly inhibits slanDC-induced proliferation and differentiation of CD4(+) T cells. In addition, the capacity of slanDCs to promote interferon-gamma secretion and tumor-directed cytotoxicity of natural killer (NK) cells is markedly impaired by bortezomib. These results provide evidence that bortezomib significantly reduces the ability of native human blood DCs to regulate innate and adaptive anti-tumor immunity and may have implications for the design of therapeutic strategies combining DC vaccination and bortezomib treatment.

Human Peripheral Blood Dendritic Cells and Monocyte Subsets Display Similar Chemokine Receptor Expression Profiles with Differential Migratory Responses

Human antigen presenting cells (APC) found in peripheral blood are considered to be precursors that have been released from the bone marrow and are in transit to the peripheral tissues. These APC populations include myeloid dendritic cells (mDC), plasmacytoid DC (pDC) and monocytes (Mo). To assign specialized functional roles and stages of development for APCs, CD33 expressing APC subsets were examined for their capacity to respond to chemokines. Three major CD33(+) subsets including CD33(bright)CD14(bright) Mo, CD33(bright)CD14(-) CD11c(+) mDC and CD33(dim)CD14(-) pDC were present. Dendritic cells subsets and Mo expressed low levels of CC and CXC receptors, but distinctive chemokine receptor expression profiles were not observed. The percentage of cells expressing a particular chemokine receptor varied from donor to donor and over time in the same donor. Myeloid DC and Mo but not pDC migrated toward CXCL12 in a concentration dependent manner. Monocytes and pDC, but not myeloid DC, were attracted by high concentrations of CXCL10. All CD33(+) subsets migrated in a concentration dependent manner toward CCL19, but responded less robustly to CCL21. CCL20 was not chemoattractant for any population. Despite the finding that APC did not exhibit unique surface chemokine receptor expression patterns, they exhibited differential migration to CXCL12, CXCL10 and CCL21 but not to CCL20 or CCL19.

Induction of anti-HBs in HB vaccine nonresponders in vivo by hepatitis B surface antigen-pulsed blood dendritic cells

Antigen-pulsed dendritic cells (DCs) are now used for treatment of patients with cancers, however, the efficacy of these DCs has never been evaluated for prophylactic purposes. The aim of this study was (1) to prepare hepatitis B surface antigen (HBsAg)-pulsed human blood DCs, (2) to assess immunogenicity of HBsAg-pulsed DCs in vitro and (3) to evaluate the efficacy of HBsAg-pulsed DCs in hepatitis B (HB) vaccine nonresponders. Human peripheral blood DCs were cultured with HBsAg to prepare HBsAg-pulsed DCs. The expression of immunogenic epitopes of HBsAg on HBsAg-pulsed DCs was assessed in vitro. Finally, HBsAg-pulsed DCs were administered, intradermally to six HB vaccine nonresponders and the levels of antibody to HBsAg (anti-HBs) in the sera were assessed. HB vaccine nonresponders did not exhibit features of immediate, early or delayed adverse reactions due to administration of HBsAg-pulsed DCs. Anti-HBs were detected in the sera of all HB vaccine nonresponders within 28 days after administration of HBsAg-pulsed DCs. This study opens a new field of application of antigen-pulsed DCs for prophylactic purposes when adequate levels of protective antibody cannot be induced by traditional vaccination approaches.

Receptor-type protein tyrosine phosphatase gamma (PTPγ), a new identifier for myeloid dendritic cells and specialized macrophages

AbstractProtein tyrosine phosphatase (PTPγ) is a receptor-like molecule with a known role in murine hematopoiesis. We analyzed the regulation of PTPγ expression in the human hematopoietic system, where it was detected in human peripheral blood monocytes and dendritic cells (DCs) of myeloid and plasmacytoid phenotypes. Its expression was maintained during in vitro monocyte differentiation to dendritic cells (moDC) and was further increased after maturation with bacterial lipopolysaccharide (LPS), CD40L, and TNFα. But PTPγ was absent when monocytes from the same donor were induced to differentiate in macrophages. B and T lymphocytes did not express PTPγ. Rather, PTPγ mRNA was expressed in primary and secondary lymphoid tissues, and the highest expression was in the spleen. PTPγ was detected by immunohistochemistry in subsets of myeloid-derived DCs and specialized macrophages (tingible bodies, sinus and alveolar macrophages). Classic macrophages in infective or reactive granulomatous reactions did not express PTPγ. Increased PTPγ expression was associated with a decreased ability to induce proliferation and interferon-γ secretion in T cells by moDCs from patients with advanced pancreatic cancer. Taken together, these results indicate that PTPγ is a finely regulated protein in DC and macrophage subsets in vitro and in vivo.

Imatinib mesylate does not impair the immunogenicity of human myeloid blood dendritic cells

Imatinib mesylate does not impair the immunogenicity of human myeloid blood dendritic cells

Human 6-Sulfo LacNAc-Expressing Dendritic Cells Are Principal Producers of Early Interleukin-12 and Are Controlled by Erythrocytes

Early and high-level production of IL-12 is crucial for effective immune responses against pathogens. Up until now, the cells providing this initial IL-12 have remained elusive. Here we show that a subset of human blood dendritic cells (DC) is the principal and primary source of IL-12p70 when blood leukocytes are stimulated with the TLR4-ligand LPS or with CD40-ligand. These so-called slanDC are characterized by the 6-sulfo LacNAc modification of PSGL-1, which is identified by the mAb M-DC8. The IL-12 response of slanDC requires a few hours of in vitro maturation, which is completely blocked in the presence of erythrocytes. This inhibition of maturation depends on the expression of CD47 on erythrocytes and of its ligand SIRPalpha on DC. While strictly controlled in the blood by erythrocytes, the high IL-12- and TNF-alpha-producing capacity of slanDC in tissues may be critical in fighting off pathogens; if uncontrolled, it may lead to adverse inflammatory reactions.

Human Dendritic Cell Interactions with Whole Recombinant Yeast: Implications for HIV-1 Vaccine Development

Defects in number and function of dendritic cells (DCs) have been observed during HIV-1 infection, so therapeutic HIV-1 vaccine approaches that target or activate DCs may improve vaccine immunogenicity. To determine the potential of recombinant Saccharomyces cerevisiae yeast as an HIV-1 vaccine, we investigated interactions between yeast and human DCs. Yeast induced direct phenotypic maturation of monocyte-derived DCs (MDDCs) and enriched blood myeloid DCs (mDCs), but only indirectly matured blood plasmacytoid DCs (pDCs). Yeast-pulsed MDDCs and blood mDCs produced inflammatory cytokines and stimulated strong allo-reactive T cell proliferation. Both blood DC subsets internalized yeast, and when pulsed with yeast recombinant for HIV-1 Gag protein, both stimulated in vitro expansion of Gag-specific CD8+ memory T cells. These results suggest that S. cerevisiae yeast have potent adjuvant effects on human DCs. Furthermore, recombinant yeast-derived antigens are processed by human blood DCs for MHC class-I cross-presentation. These DC-targeting characteristics of yeast suggest that it may be an effective vaccine vector for induction of HIV-1-specific cellular immune responses.

Upon viral exposure, myeloid and plasmacytoid dendritic cells produce 3 waves of distinct chemokines to recruit immune effectors

AbstractHost response to viral infection involves distinct effectors of innate and adaptive immunity, whose mobilization needs to be coordinated to ensure protection. Here we show that influenza virus triggers, in human blood dendritic-cell (DC) subsets (ie, plasmacytoid and myeloid DCs), a coordinated chemokine (CK) secretion program with 3 successive waves. The first one, occurring at early time points (2 to 4 hours), includes CKs potentially attracting effector cells such as neutrophils, cytotoxic T cells, and natural killer (NK) cells (CXCL16, CXCL1, CXCL2, and CXCL3). The second one occurs within 8 to 12 hours and includes CKs attracting effector memory T cells (CXCL8, CCL3, CCL4, CCL5, CXCL9, CXCL10, and CXCL11). The third wave, which occurs after 24 to 48 hours, when DCs have reached the lymphoid organs, includes CCL19, CCL22, and CXCL13, which attract naive T and B lymphocytes. Thus, human blood DC subsets carry a common program of CK production, which allows for a coordinated attraction of the different immune effectors in response to viral infection.

Nonviral transfection of human umbilical cord blood dendritic cells is feasible, but the yield of dendritic cells with transgene expression limits the application of this method in cancer immunotherapy.

Dendritic cells (DC) generated from human umbilical cord blood might replace patients' DC in attempts to elicit tumor-specific immune response in cancer patients. We studied the efficiency of transfection of human cord blood DC with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene, to test if nonviral gene transfer would be a method to load DC with protein antigens for immunotherapy purposes. Cord blood mononuclear cells were cultured in serum-free medium in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF), stem cell factor (SCF) and Flt-3 ligand (FL), to generate DC from their precursors, and thereafter transfected by electroporation. Maturation of DC was induced by stimulation with GM-CSF, SCF, FL and phorbol myristate acetate (PMA). Transfected DC strongly expressed EGFP, but transfection efficiency of DC, defined as HLA-DR(+) cells lacking lineage-specific markers, did not exceed 2.5%. Expression of the reporter gene was also demonstrated in the DC generated from transfected, purified CD34(+) cord blood cells, by stimulation with GM-CSF, SCF, FL, and tumor necrosis factor alpha (TNF-alpha). Transfection of CD34(+) cells was very efficient, but proliferation of the transfected cells was much reduced as compared to the untransfected cells. Therefore, the yield of transgene-expressing DC was relatively low. In conclusion, nonviral transfection of cord blood DC proved feasible, but considering the requirements for immunotherapy in cancer patients, transfection of differentiated DC or generation of DC from transfected hematopoietic stem cells provide only a limited number of DC expressing the transgene.

Chemokine regulation in response to beryllium exposure in human peripheral blood mononuclear and dendritic cells

Exposure to beryllium (Be) induces a delayed-type hypersensitivity immune reaction in the lungs of susceptible individuals, which leads to the onset of Be sensitivity and Chronic Beryllium Disease (CBD). Although some mechanistic aspects of CBD have begun to be characterized, very little is known about the molecular mechanisms by which Be activates the host immune response. To gain insight into the cellular response to Be exposure, we have performed global microarray analysis using a mixture of peripheral blood mononuclear and dendritic cells (PBMC/DCs) from a non-CBD source to identify genes that are specifically upregulated in response to BeSO 4 stimulation, compared to a control metal salt, Al 2(SO 4) 3. We identified a number of upregulated immunomodulatory genes, including several chemokines in the MIP-1 and GRO families. Using PBMC/DCs from three different donors, we demonstrate that BeSO 4 stimulation generally exhibits an increased rate of both chemokine mRNA transcription and release compared to Al 2(SO 4) 3 exposure, although variations among the individual donors do exist. We show that MIP-1α and MIP-1β neutralizing antibodies can partially inhibit the ability of BeSO 4 to stimulate cell migration of PBMC/DCs in vitro. Finally, incubation of PBMC/DCs with BeSO 4 altered the binding of the transcription factor RUNX to the MIP-1α promoter consensus sequence, indicating that Be can regulate chemokine gene activation. Taken together, these results suggest a model in which Be stimulation of PBMC/DCs can modulate the expression and release of different chemokines, leading to the migration of lymphocytes to the lung and the formation of a localized environment for development of Be disease in susceptible individuals.

Human BDCA-1 Positive Blood Dendritic Cells in Immunosuppressed Patients: Phenotype, Function and Maturation.

Immunosuppressive drugs used in patients (pts) after stem cell / organ transplantation (Tx) as well as in pts with autoimmune disease are known to impair the cellular immune response. This results in an increased incidence of viral infections and viral associated malignancies which has been ascribed to the effect of immunosuppressive drugs on lymphocytes. However, in vitro data indicate that immunosuppressive drugs also target Dendritic Cells (DCs), the most potent antigen-presenting cells and initiators of lymphocyte responses. So far, most studies are based on in vitro data obtained with DC culture in the presence of different concentrations of single immunosuppressive drugs. To investigate the effect of immunosuppression on DC phenotype and function in vivo, we quantitatively and qualitatively analyzed freshly isolated human BDCA-1(CD1c) positive DCs from 15 solid organ transplant (SOT) recipients under immunosuppressive treatment. The percentage of BDCA-1 positive cells among total PBMCs was not statistically different in pts vs ctrls (0,52 vs 0,65, p<0,18). BDCA-1 positive DCs were analyzed for expression of HLA class I and II, CD14, costimmulatory molecules and chemokine expression. Interestingly, CD14 was found to be significantly higher expressed on pt-DCs vs ctrl-DCs suggesting a more immature DC-phenotype. We observed a trend toward a reduced expression of HLA-DR and CD86 on pts-DCs as compared to ctrls-DCs (p=0,059). Surface profile of BDCA-1 positive DCs was also analyzed after 48h of LPS and CD40L stimulation. Here we found a marked upregulation of HLA-DR and CD86 in pts- DCs as well as ctrl-DCs. Supernatant of stimulated DCs was analyzed with cytokine capture beads for secretion of inflammatory cytokines. High secretion of IL-6, IL-1 beta and partially of TNF-alpha by stimulated DCs was observed in both groups. Other Th2 type cytokines (IL-10, IL-4, IL-5) and Th1 type cytokines like IFN-gamma and Il-2 were not significantly secreted. We additionally addressed the question if mature and functionally competent DCs could be generated ex vivo from this pts cohort. After 9 days of culture with GM-CSF, IL-4, IL-1, IL-6, TNF-alpha and PGE2 fully mature DCs could be generated. Co-culture of EBV-peptide-pulsed DCs with autologous T-cells resulted in significant expansion of EBV-specific T cells in pts and ctrls. These T cells were fully functional as shown by IFN-γ secretion detected by ELISPOT. In summary, this is the first analysis of freshly isolated BDCA-1 positive DCs from immunosuppressed pts. Our data support the notion that immunosuppressive drugs target DCs and contribute to a maturation defect of circulating blood DCs which may help to understand the mechanism of impaired cellular immune responses in immunosuppressed pts. However, ex vivo generated DCs from immunosuppressed pts do not show an impairment in phenotype and function, suggesting that they could be efficiently be used in immunotherapeutic strategies.

TGF-β1, in Synergy with M-CSF Induce Generation of a Novel Type of Human Cord Blood Dendritic Cell That Is Both Anti-Inflammatory and Capable of Ex-Vivo Expansion of Myeloid Progenitor Cells.

Tolerogenic dendritic cells (DCs) could be a promising cellular therapeutic tool in human transplantation due to their potential for silencing unwanted immune reactions. However, it is not known whether tolerogenic DCs are capable of regulating human hematopoiesis. We recently reported that M-CSF, in combination with IL-4, induced cord blood monocyte differentiation into a population of dendritic cells (DC) with tolerogenic potential (M-DC) (Li et.al. J. Immol. 174: 4706, 2005.). TGF-β1 has potent immune regulatory functions on DCs. In this context, we investigated effects of TGF-β1 on cord blood M-DC differentiation by characterizing phenotype, cytokine production and allogeneic mixed lymphocyte reaction (MLR) of the resulting DC population (TGF/M-DC). We also evaluated effects of the resultant cells on expansion and growth factor withdrawal-induced apoptosis of human CD34+ progenitors isolated from cord blood. The effects were compared to those of TGF-β1, GM-CSF and IL-4-induced DCs (TGF/DC). Addition of TGF-β1 to highly purified cord blood monocytes cultured with M-CSF and IL-4 changed M-DC morphology to that of a homogeneous DC morphology (TGF/M-DC). Similar to M-DC, TGF/M-DC negatively stained for monocyte/macrophage markers CD14 and CD16, and positively stained for CD86 and HLA-DR, molecules involved in antigen presentation. It appeared that TGF/M-DC were bona fide DC with typical DC morphology and phenotype. In comparison with TGF-DC, TGF/M-DC produced high amounts of IL-10, but displayed reduced capacity in induction of allogeneic MLR, features categorizing them more appropriately into an anti-inflammatory DC subset. We generated conditioned medium (CM) from both types of DC by stimulation of these DC with LPS and studied effects of their CM on ex-vivo expansion of CD34+ progenitors isolated from cord blood. In the absence of DC CM, the combination of SCF, Flt3 ligand and TPO (SFT, a cytokine combination widely used for cell expansion) induced a 10.5 ± 1.8 fold expansion of myeloid progenitors (CFU-GM) in 1 week culture. TGF/M-DC CM (25% v/v), but not TGF DC CM, in synergy with SFT further expanded myeloid progenitors (CFU-GM) 19.4 ± 2.7 fold beyond that seen with SFT alone. The effects of TGF/M-DC CM was accompanied with a significant increase of CD34+ cell numbers after 1 week (TGF/M-DC CM in combination with SFT, TGF-DC CM in combination with SFT, and SFT alone expanded CD34+ cells by 8.4 ± 2.8 fold, 3.8 ± 2.8 fold and 5.9 ± 1.1 fold respectively). TGF/M-DC CM and TGF/DC CM prevented growth factor withdrawal-induced apoptosis of CD34+ cells, as analyzed by annexin-V staining. Therefore, TGF/M-DC might be capable of in vivo silencing of unwanted immune responses while promoting myeloid recovery. These features of TGF/M-DC make them an interesting cellular therapeutic candidate to potentially facilitate hematopoietic recovery as well as down-modulate unwanted immune responses after hematopoietic stem cell transplantation.

Ex Vivo Expanded Dendritic Cells Home to T-Cell Zones of Lymphoid Organs and Survive in Vivo after Allogeneic Bone Marrow Transplantation

Little is known about adoptive transfer of allogeneic ex vivo expanded dendritic cells (eDCs). We investigated the trafficking pattern of eDCs in mice after allogeneic bone marrow transplantation by using bioluminescence imaging. eDCs were expanded from bone marrow precursors in the presence of GM-CSF, interleukin-4, and Flt3L and retrovirally transduced to express luciferase (luc) and green fluorescence protein (gfp). Flow cytometry showed polyclonal DC populations after expansion that consisted of CD11c+CD11b+ and CD11c-CD11b+ cells that co-expressed CD40, CD80, CD86, and MHCII. eDCs were functional in mixed lymphocyte reactions and produced tumor necrosis factor-alpha on phytohemagglutinin stimulation. The eDCs were then injected intravenously into BALB/c recipient mice that had received allogeneic bone marrow transplantation 6 weeks previously. On day 1 after transfer, eDCs were detected by bioluminescence imaging throughout the lungs and spleen. In the later course, signals were observed throughout thymus, lower abdomen, and spleen throughout a period of more than 42 days. Immunofluorescence microscopy confirmed CD11c positivity on the gfp+ donor cells, which localized in T-cell zones of mesenteric lymph nodes, Peyer's patches, spleen, and thymus. These findings are important for adoptive immunotherapies because they indicate that eDCs migrate efficiently in vivo and are capable of surviving long term.

Differential Processing of Autoantigens in Lysosomes from Human Monocyte-Derived and Peripheral Blood Dendritic Cells

Dendritic cells (DC) initiate immunity and maintain tolerance. Although in vitro-generated DC, usually derived from peripheral blood monocytes (MO-DC), serve as prototype DC to analyze the biology and biochemistry of DC, phenotypically distinct primary types of DC, including CD1c-DC, are present in peripheral blood (PB-DC). The composition of lysosomal proteases in PB-DC and the way their MHC class II-associated Ag-processing machinery handles a clinically relevant Ag are unknown. We show that CD1c-DC lack significant amounts of active cathepsins (Cat) S, L, and B as well as the asparagine-specific endopeptidase, the major enzymes believed to mediate MHC class II-associated Ag processing. However, at a functional level, lysosomal extracts from CD1c-DC processed the multiple sclerosis-associated autoantigens myelin basic protein and myelin oligodendrocyte glycoprotein in vitro more effectively than MO-DC. Although processing was dominated by CatS, CatD, and asparagine-specific endopeptidase in MO-DC, it was dominated by CatG in CD1c-DC. Thus, human MO-DC and PB-DC significantly differ with respect to their repertoire of active endocytic proteases, so that both proteolytic machineries process a given autoantigen via different proteolytic pathways.

Porphyromonas gingivalis fimbriae induce CD14+CD16+ dendritic cell phenotype via TLR2

Dendritic cells (DCs) play a critical role in activation of T cells as well as shaping immune responses. DCs express a pattern of Toll-like receptors (TLRs) to recognize invading pathogens and to initiate specific immune responses. In this study, we investigated the effect of Porphyromonas gingivalis fimbriae on the phenotype and function of human peripheral blood DCs (PBDCs) in comparison to TLR2 and TLR4 ligand, peptidoglycan (PGN) from Staphylococcus aureus and Escherichia coli O55:B5 LPS, respectively. P. gingivalis fimbriae and PGN preferentially upregulated CD14 and CD16 expression on immature PBDCs (CD14−CD16−), although E. coli LPS did not alter the expression of these molecules. Fimbriae-stimulated DCs also exhibited a different profile of cytokine production and surface molecule expression as compared with E. coli LPS-stimulated DCs. Pretreatment of PBDCs with anti-TLR2 monoclonal antibody (mAb), but not with anti-TLR4 mAb, abrogated the upregulation of CD14 and CD16 on fimbriae and PGN-treated DCs. These results indicate that different TLR signaling affects the mature DC phenotype and function and is thus crucial to the regulation of immunity to the pathogen.

Allogeneic Dendritomas Induce Anti‐tumour Immunity Against Metastatic Colon Cancer

Colon cancer (CC) is one of the most common malignancies in the Western world. Although surgical resection is the first choice worldwide, at this point an effective approach for the treatment of patients with metastasis and cancer recurrence post-operation has not yet been found. The aim of this study was to investigate the role of the allogeneic dendritomas from fusion of dendritic cells (DC) and metastatic CC cells in the activation of anti-tumour immunity against metastatic CC. Dendritomas were generated by fused allogeneic human peripheral blood DC with metastatic CC cells using 50% polyethylene glycol. The proliferation of the T cells and the toxicity of the cytotoxic T lymphocytes were observed after T cell pulsed with allogeneic dendritomas. The activated ratios of CD4+T helper 1 and CD8+Tc1 cells were about 51.55 and 65.60% after T cells were mixed with fusions for 24 h, which higher than those of controls. The proliferation of T cells were significantly higher than those of control after T cell pulsed with dendritomas (P < 0.01). Significantly, the activated CD8+ T lymphocytes effectively lysed the CC cells. These results demonstrate that allogeneic dendritomas activate T-cell responses against metastatic CC cells.

Differential capability for phagocytosis of apoptotic and necrotic leukemia cells by human peripheral blood dendritic cell subsets

CD11c+ dendritic cells (DC) and plasmacytoid DC (PDC) are the two major DC subsets in human peripheral blood. For the purpose of immunotherapy with DC, it is important to investigate the phagocytosis of killed tumor cells by different DC subsets. Using immature monocyte-derived DC (iMoDC) as reference, we have compared the ability of CD11c+ DC and PDC to phagocytose apoptotic and necrotic K562 leukemia cells. Freshly isolated CD11c+ DC phagocytosed apoptotic and necrotic K562 cells, whereas PDC did not show any evidence of uptake of dead cells. Blocking studies showed that CD36 is importantly involved in uptake of apoptotic and necrotic material. CD91 and CD11c were also involved. In addition, we found that beta5 integrin was expressed on CD11c+ DC but not in its classical association with alphaV. Uptake of apoptotic K562 cells by CD11c+ DC was increased following incubation with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4, alone or in combination with transforming growth factor-beta1, to levels comparable with those observed for iMoDC. Phagocytosis of dead cellular material by the GM-CSF/IL-4-treated CD11c+ DC was largely restricted to a subset expressing low levels of human leukocyte antigen-DR and CD83. Thus, the relationship between phagocytosis of antigenic material and expression of maturation-related cell-surface molecules is similar for CD11c+ DC and MoDC. We conclude that CD11c+ DC in peripheral blood are precursor cells, which under the influence of cytokines, differentiate to cells with DC phenotype and function.

Activated human umbilical cord blood dendritic cells kill tumor cells without damaging normal hematological progenitor cells

Apart from their role as antigen presenting cells, human peripheral blood monocyte and CD34+ cell-derived dendritic cells (DC), have been demonstrated to exert cytotoxicity against some tumor cells, and their tumoricidal activity can be enhanced by some stimili. However, there have been no reports concerning the tumoricidal activity of human cord blood dendritic cells (CBDC). In this article, we report that human cord blood monocyte-derived DC acquire the ability to kill hematological tumor cells, after activation with lipopolysaccharide (LPS) or gamma-interferon (IFN-gamma), associated with the enhanced TNF-alpha-related apoptosis-inducing ligand (TRAIL) expression in CBDC cytoplasm. The CD14-positive cells collected from cord blood were induced to CBDC in vitro. After activation with IFN-gamma for 12 h, CBDC exhibited cytotoxicity against HL60 and Jurkat cells, while activation with LPS induced cytotoxicity against Daudi and Jurkat cells. However, both LPS- and IFN-gamma-stimulated CBDC showed no cytotoxic activity against normal CD14-negative cord blood mononuclear cells. The formation of umbilical cord hematopoietic progenitor colonies, identified as burst-forming unit-erythroid and colony-forming unit granulocyte-macrophage, was not inhibited by stimulated or unstimulated CBDC. IFN-gamma or LPS stimulation enhanced intracellular but not cellular surface TRAIL, and neither intracellular nor cellular surface tumor necrosing factor-alpha and Fas Ligand as analyzed by flow cytometry. Our results show that activated CBDC can serve as cytotoxic cells against hematological tumor cells without damaging the normal hematopoietic progenitor cells.

A Three-Dimensional Cellular Model of the Human Respiratory Tract to Study the Interaction with Particles

A novel triple co-culture model of the human airway barrier was designed to simulate the cellular part of the air-blood barrier of the respiratory tract represented by macrophages, epithelial cells, and dendritic cells. When epithelial cells (A549 cells) were grown on filter inserts with pores of 3.0 mum in diameter in a two-chamber system, they formed monolayers with polarization into apical and basolateral domains. The epithelial cell cultures were then supplemented with human blood monocyte-derived macrophages and dendritic cells on the apical and basal aspect, respectively. The single-cell cultures as well as the triple co-cultures were characterized in terms of a number of typical features, for example, morphology of cell types, integrity of epithelial layer, and expression of specific cell surface markers (CD14 for macrophages and CD86 for dendritic cells). The interplay of epithelial cells with macrophages and dendritic cells during the uptake of polystyrene particles (1 mum in diameter) was investigated with confocal laser scanning and conventional transmission electron microscopy. Particles were found in all three cell types, although dendritic cells were not directly exposed to the particles. More investigations are needed to understand the translocation pathway.

Chemokine-Like Receptor 1 Expression and Chemerin-Directed Chemotaxis Distinguish Plasmacytoid from Myeloid Dendritic Cells in Human Blood

Plasmacytoid dendritic cells (pDCs) are versatile cells of the immune response, secreting type I IFNs and differentiating into potent immunogenic or tolerogenic APCs. pDCs can express adhesion and chemokine receptors for lymphoid tissues, but are also recruited by unknown mechanisms during tissue inflammation. We use a novel mAb specific for serpentine chemokine-like receptor 1 (CMKLR1) to evaluate its expression by circulating leukocytes in humans. We show that CMKLR1 is expressed by circulating pDCs in human blood, whereas myeloid DCs (mDCs) as well as lymphocytes, monocytes, neutrophils, and eosinophils are negative. We identify a major serum agonist activity for CMKLR1 as chemerin, a proteolytically activated attractant and the sole known ligand for CMKLR1, and we show that chemerin is activated during blood coagulation and attracts pDC but not mDC in ex vivo chemotaxis assays. We conclude that CMKLR1 expression and chemerin-mediated chemotaxis distinguish circulating pDCs from mDCs, providing a potential mechanism for their differential contribution to or regulation of immune responses at sites of bleeding or inflammatory protease activity.