Human Peripheral Blood Dendritic Cells Research Articles

HIV-1 infection of human peripheral blood, cord blood and tonsillar dendritic cells

The role of peripheral blood and tissue dendritic cells (DC) in the pathogenesis of human immunodeficiency virus (HIV) infection has been studied by several groups of investigators. The ability of DC to support productive HIV infection in vitro remains controversial. As reported here, low-level replication of HIV occurs in cord blood DC (CBDC), while tonsillar DC (TDC) fail to support HIV replication. It is unclear whether peripheral blood dendritic cells (PBDC) are depleted or dysfunctional in HIV-infected individuals. While several previous reports and our own data indicate that T-cell proliferation to mitogens and antigens is reduced in the presence of HIV-exposed PBDC, the exact mechanisms involved have not been elucidated. DC appear to have the ability to transfer HIV to CD4 T-cells during an immune response. This transfer leads to productive infection and apoptotic cell death. The role of DC in the pathogenesis of HIV infection requires further definition. The availability of methods for generation of DC from cord or adult peripheral blood and for improved identification of PBDC should prove to be useful in this regard.

Cytokine expression by human peripheral blood dendritic cells stimulated in vitro with HIV-1 and herpes simplex virus.

Dendritic cells are potent stimulators of Ag-specific T cell responses and have been implicated in the pathogenesis of HIV-1 and other viral infections. Although cytokines may be involved in both of these processes, there is little information on the expression of cytokines by human blood dendritic cells. We characterized cytokine gene and protein expression in dendritic cells that were purified from normal human PBMC by flow cytometry and stimulated in vitro for up to 24 h with HIV-1 or herpes simplex virus (HSV). The unstimulated, uncultured dendritic cells were defined by their phenotype (HLA DR+ CD3- CD19- CD16- CD56- CD14-) and distinct morphology, lack of mRNA expression for CD3, CD14 and CD19, and presence of mRNA for CD4 and CD83. The purified dendritic cells also expressed CD4 (70-90%), CD33 (36-48%), and CD11c (44-54%); lacked CD1a expression (<1%); and were potent stimulators of an allogeneic MLR. The stimulated dendritic cells expressed mRNA for IFN-alpha, IL-1alpha, IL-1beta, IL-6, IL-10, IL-12, GM-CSF, and TNF-alpha within 4 to 12 h as determined by reverse transcription-PCR, with higher levels induced by HSV compared with HIV-1 strains IIIb or BaL. In contrast, the dendritic cells produced detectable protein only for IFN-alpha and IL-6 in response to HIV-1 or HSV, and IL-1beta in response to HSV within 24 h. There were no differences in expression of CD80 and CD86 surface molecules by dendritic cells that were either mock stimulated or stimulated with HIV-1 or HSV for 24 h. Production of IFN-alpha, IL-1beta, and IL-6 by dendritic cells may be important to the immunologic function of these cells and their role in the pathogenesis of HIV-1 and HSV infections.

IDENTIFICATION OF A NOVEL DENDRITIC CELL SURFACE ANTIGEN DEFINED BY CARBOHYDRATE SPECIFIC CD24 ANTIBODY CROSS‐REACTIVITY

Dendritic cells (DC) are characterized as leucocytes that lack mature lineage specific markers and stimulate naive T-lymphocyte proliferation in vitro and in vivo. The mouse heat stable antigen (HSA) participates in T lymphocyte co-stimulation and is expressed by DC isolated from thymus, skin and spleen. The human HSA homologue, CD24, is predominantly expressed by B lymphocytes and granulocytes, but its expression on DC has not been studied in detail. CD24 clearly participates in B-lymphocyte signalling but co-stimulatory activity for T lymphocytes has not yet been described. We have examined the expression of CD24 on human peripheral blood DC populations isolated directly or following in vitro culture. The CD24 antigen was absent from blood DC however, cross-reactive sialylated carbohydrate epitopes were detected on DC with some CD24 monoclonal antibodies (mAb). These CD24 mAb define a protein surface antigen, which is expressed by an immature or resting subpopulation of peripheral blood DC and is down-regulated following activation differentiation in vitro.

Functional expression of minor histocompatibility antigens on human peripheral blood dendritic cells and epidermal Langerhans cells

Adequate presentation and cell surface expression of foreign minor histocompatibility antigens (mHag) to allogeneic T cells can lead to graft-versus-host disease (GvHD) after HLA matched bone marrow transplantation (BMT). Cells of the dendritic cell (DC) lineage, including epidermal Langerhans cells (LC), are the most potent inducers of primary alloreactive T cell responses in vivo and in vitro. To explore the possible role of peripheral blood DC and of skin derived LC in the induction of alloimmune responses against mHag, we analysed the functional expression of mHag on these professional antigen-presenting cells (APC). To this end, cytotoxic T cell (CTL) clones specific for mHag H-Y and HA-1 to HA-4 were used to demonstrate the presence of these antigens on highly purified DC and LC. Our results demonstrated that, like other cells of the hepatopoietic lineage, DC and LC express all the mHag tested for. The functional expression of mHag on these potent APC suggests their involvement in the induction of mHag specific GvH directed T cell responses after allogeneic BMT.

Interleukin‐10 differentially regulates B7‐1 (CD80) and B7‐2 (CD86) expression on human peripheral blood dendritic cells

Most of the immunosuppressive effects of interleukin-10 (IL-10) are related to functional inhibition of antigen-presenting cells (APC). Herein, we investigate the influence of recombinant (r)IL-10 on human dendritic cells (DC) purified from peripheral blood of healthy volunteers. First, we found that rIL-10 inhibited in a dose-dependent manner the proliferative responses as well as the production of IL-2 and interferon-gamma (IFN-gamma) in mixed lymphocyte reaction (MLR) between purified T cells and DC. This rIL-10 effect could be attributed to a direct effect on DC, as DC preincubated with rIL-10 were found to be deficient in the induction of alloreactive T cells even when anti-IL-10 neutralizing mAb was added at the time of MLR. Flow cytometric analysis indicated that rIL-10 did not modify the expression of ICAM-1 (CD54) and B7-1 (CD80), but decreased HLA-DR and B7-2 (CD86) expression at the DC surface. We conclude that the inhibitory effect of rIL-10 on primary alloreactive T cell responses involves down-regulation of class II MHC and B7-2 expression at the DC surface.

Activation of human peripheral blood dendritic cells induces the CD86 co-stimulatory molecule.

Maximal T lymphocyte responses require presentation of antigen by major histocompatibility complex molecules and delivery of one or more co-stimulatory signals. Interaction of the CD28 molecule on T lymphocytes with its ligands on antigen-presenting cells (APC) initiates a critical co-stimulatory pathway inducing T lymphocyte proliferation and cytokine secretion. Dendritic cells (DC) are potent APC for a primary T lymphocyte response and potential CD28/CTLA-4 ligands on DC are, therefore, of particular functional relevance. In these experiments, the expression and function of the CD28/CTLA-4 ligands B7.1 (CD80) and B7.2 (CD86) were examined on human blood DC. Resting DC populations directly isolated by immunodepletion of lineage marker-positive cells lacked cell membrane expression of CD80 and expressed little or no CD86, although CD86, but not CD80 mRNA was detected by reverse transcription-polymerase chain reaction analysis. In contrast, low-density DC isolated after culture in vitro strongly expressed CD86 surface protein, but expressed limited or no CD80, although mRNA for both molecules were detected. Short-term culture of directly isolated DC up-regulated both CD80 and CD86 expression. Analysis of the kinetics of CD28/CTLA-4 ligand induction showed that surface CD86 was present within 8 h, whereas CD80 antigen was first detected after 24 h of culture. The functional importance of CD28/CTLA-4 ligand up-regulation on DC during T lymphocyte interactions was demonstrated by the ability of both CTLA-4Ig and CD86 monoclonal antibodies (mAb), but not CD80 mAb, to block an allogeneic mixed lymphocyte reaction stimulated by DC populations initially negative for CD80 and CD86. These results demonstrate that CD86 is both the earliest and functionally the predominant co-stimulatory CD28/CTLA-4 ligand on DC.

Isolation and characteristics of dendritic cell progenitors from the bone marrow of the Hodgkin's disease patients.

It has been previously shown that GM-CSF promotes differentiation and survival of human peripheral blood dendritic cells (DC) in vitro1. Human bone marrow progenitors of dendritic cells were identified in semi-solid cultures supplemented with conditioned media from PHA-stimulated leukocytes2. GM-CSF and TNF-α have been shown to cooperate in the regulation of DC growth from CD34+ progenitors in vitro3,4,5.KeywordsDendntlc CellBone Marrow Mononuclear CellBone Marrow ProgenitorHematopoietic Cell LineageNodular scleroSISThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

IL-10 inhibits the primary allogeneic T cell response to human peripheral blood dendritic cells.

Most of the immunosuppressive properties of IL-10 are related to its action on antigen presenting cells (APC). Until now, several mechanisms have been described by which IL-10 inhibits the ability of monocytes/macrophages to induce T cell activation. Indeed, IL-10 down-regulates their expression of MHC class II molecules1 as well as of ICAM-1 and B7-1 accessory molecules2,3. Moreover, the inhibition by IL-10 of IL-12 and IL-1 synthesis by monocytes was shown to be responsible for the inhibition of IFN-y production by peripheral blood mononuclear cells (PBMC)4. As far as the effects of IL-10 on dendritic cells are concerned, IL-10 was shown to inhibit the ability of mouse splenic dendritic cells to induce interferon (IFN)-y production by antigen-specific TH1 clones or alloreactive splenic T cells5. In addition, human epidermal Langerhans cells preincubated with IL-10 were found to be deficient in the triggering of a mixed lymphocyte reaction6. As a first approach to study the effects of IL-10 on human peripheral blood dendritic cells (PBDC), we analyzed the influence of IL-10 on a primary allogeneic T cell response induced by those cells.

Human peripheral blood dendritic cell subsets. Isolation and characterization of precursor and mature antigen-presenting cells.

Dendritic cells (DC) are the major APC capable of stimulating resting T cells in human peripheral blood (PB). Recent evidence suggested that various subsets of DC and monocytes might circulate in human PB, but their exact phenotype and function had not been delineated. We have previously characterized a population of human PB DC precursors that express the myeloid marker CD33, but not the monocyte marker CD14. To identify and characterize further functional myeloid APC subsets, triple color FACS analysis and sorting was used. A CD33dimCD14dimCD16+ monocyte subset, with similar APC function but less efficient accessory function than CD14bright monocytes, was isolated. In addition to the CD33+CD14dimCD16- DC precursors, a smaller population (0.1 to 0.2% of PBMC) of CD33brightCD14dimCD16- cells with potent APC function was identified. This DC population expressed greater amounts of MHC class II, adhesion, and accessory molecules, and demonstrated a greater costimulatory capacity when freshly isolated than CD33dimCD14dim DC precursors, and therefore had the characteristics of mature, possibly tissue-derived DC. When freshly isolated, however, these DC did not express B7, and up-regulation of accessory function occurred after in vitro differentiation. These data demonstrate multiple circulating myeloid accessory and APC subsets in human PB. Phenotypic and functional differences suggest that they are at different stages of differentiation, and have specialized roles in Ag presentation in vivo. Furthermore, full functional DC differentiation, associated with B7 expression and the capacity to activate T cells maximally, is likely to occur only in specific physiologic circumstances.

Generation of antigen-specific CD8+ CTLs from naive precursors.

Class I MHC-restricted CTLs are an important component of the host immune response against viral infections, and CTL effectors can often be isolated from infected individuals. However, the mechanism responsible for the induction of CTLs is incompletely understood because, in part, of the difficulty in generating such cells in vitro from naive precursors. In the present study we have used human peripheral blood dendritic cells (DCs), devoid of CD4+ T cells, to sensitize naive CD8+ T cells to exogenous Ags, resulting in the generation of Ag specific CTL effectors. With this system, Ag-specific CTL lines were generated to a complex glycoprotein, keyhole limpet hemocyanin, and to multiple small (9-15 amino acids) synthetic peptides derived from conserved regions of the HIV-1 gag and envelope proteins. The HIV-1-specific CTLs demonstrated potent HLA class I restricted killing of both Ag pulsed and virally infected target cells. In contrast to Ag-pulsed DCs, Ag-pulsed monocytes failed to sensitize CTL precursors although they could be used as feeders for purposes of CTL expansion and as target cells in cytolytic assays. With the use of the system described herein, a detailed analysis of the primary human T cell response to foreign Ags is now feasible, and CTL of desired specificity can be generated for potential clinical use in adoptive immunotherapy protocols.

Comparative accessory cell function of human peripheral blood dendritic cells and monocytes.

The capacity of human peripheral blood (PB) dendritic cells (DC) and monocytes to facilitate T cell activation and the interaction molecules employed were compared. We have shown that precursors of DC constitute 2 to 3% of circulating PBMC, and can be isolated as CD33+CD14dim cells, whereas monocytes are CD33+CD14bright. Freshly obtained DC expressed similar densities of HLA-DR and the accessory molecules LFA-3, ICAM-1, and B7 as monocytes; after a 36-h incubation the expression of HLA-DR, ICAM-1, and B7 increased on both APC. Accessory cell function was examined in PB T cell cultures stimulated by suboptimal concentrations of immobilized mAb to CD3, and by stimulation of an allospecific T cell line. Freshly isolated monocytes and DC were comparable accessory cells in these assays, but their accessory function was increased by in vitro preincubation, although cultured DC and monocytes were comparably active. In contrast, DC were much more effective stimulators of freshly isolated allogeneic T cells than monocytes. DC were much more effective stimulators of freshly isolated allogeneic PB CD4+ naive and memory T cells than monocytes, whereas DC and monocytes were comparable accessory cells for memory and naive T cells stimulated with immobilized anti-CD3. The accessory molecules ICAM-1, LFA-3, and B7 were used comparably by DC and monocytes for accessory function in the presence of immobilized anti-CD3 and in the MLR, and none was unique for either APC population. These accessory molecules costimulated T cells in an additive fashion. Although immature blood DC and monocytes expressed minimal B7 and did not utilize it as an accessory molecule, B7 played an important role in the increased accessory function of differentiated APC. The results indicate that PB DC and monocytes function most efficiently after differentiation into mature cells that express increased amounts of MHC and other accessory molecules. Because DC and monocytes exhibit comparable accessory function in anti-CD3 T cell stimulation, differences in the expression of MHC molecules and/or their bound peptides are likely to explain the markedly enhanced capacity of DC to stimulate allogeneic PB T cells.

Isolation and characterization of human peripheral blood dendritic cells.

Dendritic cells (DC) are potent APC that can be purified from cultured peripheral blood non-T cells. Because no specific cell surface marker has been found, the lineage of DC remains obscure. The purpose of these studies was to determine the circulating blood cells that could give rise to functional human DC. DC were enriched when purified by standard techniques from non-T cells that were treated with L-leucyl-L-leucine methyl ester, known to be toxic to monocytes and cytolytic cells. To determine whether monocytes or B cells could give rise to DC, fresh non-T cells were sorted into CD14+ monocytes, CD19+ B cells, and CD14- and CD19- cells. Although there was some enrichment for APC function by cultured nonadherent CD14- or CD19- cells, a marked enrichment for cells with dendritic morphology and potent APC function was found in the population that was sorted by the absence of expression of CD14, CD19, CD3, and CD16. More than 90% of the CD14-CD19-CD16-CD3- sorted cells, and of control DC, expressed the myeloid markers CD13 and CD33. Therefore, fresh non-T cells were sorted based on the expression of these myeloid markers. In comparison with CD33-CD14- B cells, some of the CD33+ cells expressed CD14 dimly. However, they were easily distinguished from monocytes, which intensely expressed CD14. CD33+CD14dim cells developed dendritic processes and were more potent APC than control DC, CD33+CD14+, or CD33-CD14- cells. Although freshly isolated CD33+CD14dim DC expressed a number of cell surface molecules also expressed by CD14+ monocytes, they demonstrated lower levels of lysosomal enzymes and a lack of FcR-mediated phagocytosis in comparison with monocytes. Differentiation of morphology and phenotype of CD33+CD14dim cells occurred within 6 to 36 h in culture. However, the CD33+CD14dim cells could effectively function as APC without prolonged preincubation to develop dendritic morphology. These data indicate that human blood DC arise from precursors that express the myeloid lineage markers CD13 and CD33, but are functionally distinct from classic CD14+ monocytes.

Human Peripheral Blood Dendritic Cells Concentrate in Contrast to Monocytes Intracellular HLA Class II Molecules in a Juxtanuclear Position

The localization of histocompatibility antigens of the HLA-D locus in dendritic cells (DC) and monocytes (Mo) isolated from human peripheral blood was investigated. Functionally DC were characterized by their capacity to act as strong stimulators in an allogeneic mixed leucocyte reaction.In cytospins, DC were differentiated from Mo by dendritic morphology, strong HLA class II and moderate RFD 1 expression on the plasma membrane and acid phosphatase activity in a juxtanuclear spot.Ultrathin cryosections showed that DC had a heavily labelled plasma membrane for HLA-D.In addition, these antigens were found in intracellular vesicles predominantly located in the juxtanuclear zone.This pattern of labelling was not seen in Mo.Obviously, DC concentrate intracellular class II antigens in the same area as lysosomal activity.These results may indicate that this juxtanuclear area is a center of antigen processing in DC.

Granulocyte-macrophage colony-stimulating factor promotes differentiation and survival of human peripheral blood dendritic cells in vitro.

Interest in human dendritic cells (DC) has been heightened recently by the discovery that this cell type is a primary target of the human immunodeficiency virus, the causative agent of AIDS. DC are bone marrow-derived cells with an extraordinarily potent ability to promote the immunological activity of T lymphocytes. Unfortunately, since DC constitute less than 0.5% of peripheral blood mononuclear cells and die within a few days of their isolation, they are not readily accessible to study. We report here that granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine with well-recognized effects on granulocyte and macrophage maturation, profoundly affects the morphology and viability of DC isolated from peripheral blood. GM-CSF not only promotes DC survival but also induces DC differentiation to mobile, reversibly adherent cells with long-branched projections. DC cultured in GM-CSF survive for up to 6 wk and retain their ability to stimulate the proliferation of T cells in allogeneic and autologous mixed leukocyte reactions.

Effect of anti-CD4, anti-OKDR antibody and γ-interferon on cluster formation by human peripheral blood dendritic cells and T cells

Dendritic cells (DC) were successfully isolated from human peripheral blood and investigated for cluster formation by co-culturing them with autologous T cells at 37°C.Cluster formation was recognized in the presense of anti-CD4, anti-OKDR antibody and γ-interferon (IFN). However, the proliferative response of clusters as measured by 3H-thymidine incorporation was depressed in a dose-dependent manner in the presense of anti-CD4 and anti-OKDR antibody. On the other hand, non-clustered cells showed slight 3H-thymidine incorporation in the presense or absense of anti-CD4 and anti-OKDR antibody. The proliferative response of clusters was augmented by pretreating DC with interleukin 1 (IL-1) and depressed by pretreatment with γ-IFN.These results suggested that IL-1 has a stimulatory effect on the proliferative reponse of the cluster while anti-CD4, anti-OKDR antibody and γ-IFN have an inhibitory effect, even though these factors exhibited no effect on cluster formation.

Susceptibility of human peripheral blood dendritic cells to infection by human immunodeficiency virus.

Preparations of human peripheral blood dendritic cells have been infected with human immunodeficiency virus (HIV). After 5 days in culture they were examined by electron microscopy. Virus was observed budding from the plasma membrane of dendritic cells and mature virions were observed on the cell surface. In addition, a second cell type, similar in morphology to 'classical' dendritic cells but containing numerous cytoplasmic granules, was also found to support replication of the virus. We speculate that the growth of HIV in dendritic cells could cause immunosuppression by impairing antigen presentation.