Human monocyte-derived and CD83(+) blood dendritic cells enhance NK cell-mediated cytotoxicity

Dendritic cells (DC) are known to be the most potent APC and to stimulate antigen-specific T cell responses. Recently it was reported that murine DC were also capable of modulating the innate immunity by stimulating NK cells through cell-to-cell contact. In the present study, we examined whether human DC could affect NK activity. Both monocyte-derived and CD83(+) blood DC were tested. The addition of DC to cultures of CD56(+) cells resulted in the significant dose-dependent enhancement of the killing activity against various NK-sensitive targets. The resultant activity was comparable to that induced by optimal concentrations of various cytokines, including IL-2, IL-12, IL-15 and IFN-gamma. Interestingly, DC enhanced the cytotoxicity of CD3(-)CD56(+) NK cells, but not that of CD3(+)CD56(+) T cells. Experiments using transwells clearly demonstrated that the enhancement of NK activity by DC was mediated by soluble factors produced by DC. The culture supernatants of DC also stimulated NK activity. The treatment of both DC and their supernatants with anti-human IL-12 or IL-18 antibodies did not block the enhancement of NK cell-mediated cytolysis by DC, indicating that other factor(s) produced by DC were responsible for the enhancement of NK activity. These results suggest that human myeloid DC can modulate innate immunity by enhancing NK activity.     

Diverse functional activity of CD83+ monocyte-derived dendritic cells and the implications for cancer vaccines

Antigen-loaded dendritic cells (DCs) provide key regulatory signals to T cells during a developing antitumor response. In addition to providing costimulation, mature DC provides cytokine and chemokine signals that can define the T1 vs T2 nature of the antitumor T-cell response as well as whether T cells engage in direct interactions with tumor cells. In serum-free culture conditions that hasten the differentiation of monocytes into mature DCs, certain agents, such as CD40L, accelerate phenotypic maturation (e.g., CD83 and costimulatory molecule expression) without influencing the acquisition of Dc1/Dc2 characteristics. In contrast, exposure to serum-free medium and interferon-gamma (IFN-gamma) rapidly influences CD83+ DCs to secrete high levels of IL-12, IL-6, and MIP-1beta, and promotes Dcl differentiation. In contrast, CD83+ DCs matured in serum-free medium in the absence of IFN-gamma, or in the presence of calcium signaling agents, prostaglandin-E2, or IFN-alpha, produce no IL-12, scant IL-6, and prodigious IL-8, MDC, and TARC, and promote Dc2 differentiation. T cells sensitized via IL-12-secreting, peptide-pulsed DCs secrete cytokines when subsequently exposed to relevant peptide-pulsed antigen-presenting cells (APCs) or to HLA-compatible tumor cells endogenously expressing the peptide. In contrast, T cells sensitized via IL-12 nonsecreting DC were limited to antigenic reactivation through APC contact rather than tumor cell contact. Therefore, the development of antitumor responses can be dramatically influenced not only by costimulation, but also by the cytokine and chemokine production of DCs, which must be considered in the development of cancer vaccines.     

Comparison of CD34 and monocyte-derived dendritic cells from mobilized peripheral blood from cancer patients

Dendritic cells (DCs) are potent antigen-presenting cells that are integral to the initiation of T-cell immunity. Two cell types can be used as a source for generating DCs: monocytes and CD34(+) stem cells. Despite many investigations characterizing DCs, none have performed a direct paired comparison of monocyte and stem cell-derived DCs. Therefore, it is unclear whether one cell source has particular advantages over the other, or whether inherent differences exist between the two populations. We undertook the following study to determine if there were any differences in DCs generated from monocytes or CD34(+) cells from mobilized peripheral blood. DCs were generated by culturing the adherent cells (monocytes) in interleukin-4 and GM-CSF for 7 days, or by culturing nonadherent cells (CD34(+)) in the presence of GM-CSF and tumor necrosis factor alpha for 14 days. The resulting DCs were compared morphologically, phenotypically, functionally, and by yield. We could generate morphologically and phenotypically similar DCs. Differences were encountered when expression levels of some cell surface markers were examined (CD86, HLA-DR). There was no difference in how the DCs performed in a mixed lymphocyte reaction (p = .3). Further, no statistical difference was discovered when we examined cellular (DC) yield (p = .1); however, there was a significant difference when yield was normalized to the starting number of monocytes or CD34(+) cells (p = .016). Together, these data demonstrate that differences do exist between monocyte-derived DCs and CD34-derived DCs from the same cellular product (apheresis) from the same individual.     

An in vitro evaluation of the potential suitability of peripheral blood CD14(+) and bone marrow CD34(+)-derived dendritic cells for a tolerance inducing regimen in the primate

Dendritic cells (DC) represent a tool not only for immune activation, but also potentially for tolerance induction in transplantation. This latter approach is yet to be explored in a pre-clinical primate model. Since no information concerning baboon DC has been available, we characterised the DC of this species derived in vitro from bone marrow (CD34(+)) and peripheral blood (CD14(+)) precursors to determine which would be the most suitable for a tolerance inducing strategy. Baboon DC were differentiated in vitro using protocols similar to those used in humans and their maturation status was assessed and compared according to their phenotype and function. Based on both phenotypic and functional criteria, the CD14-derived baboon DC appeared to be less mature DC, necessitating an additional stimulus in order to become fully mature. The CD34-derived DC on the other hand appeared more mature in nature, without necessarily requiring exposure to overt maturation signals. We suggest therefore that, in the baboon, peripheral blood CD14-derived DC may be more suitable for protocols where tolerance induction is the goal. We now aim to perform further in vitro investigations into the potential tolerance inducing effects of CD14-derived DC alone or in association with other strategies that would be applicable in vivo.     

Induction of various immune modulatory molecules in CD34(+) hematopoietic cells

Lipopolysaccharide (LPS) has been shown to induce proliferation of human T-lymphocytes only in the presence of monocytes and CD34(+) hematopoietic cells (HCs) from peripheral blood. This finding provided evidence of an active role of CD34(+) HCs during inflammation and immunological events. To investigate mechanisms by which CD34(+) HCs become activated and exert their immune-modulatory function, we used the human CD34(+) acute myeloid leukemia cell line KG-1a and CD34(+) bone marrow cells (BMCs). We showed that culture supernatants of LPS-stimulated mononuclear cells (SUP(LPS)) as well as tumor necrosis factor alpha (TauNF-alpha), but not LPS alone, can activate nuclear factor-kappaB in KG-1a cells. By cDNA subtraction and multiplex polymerase chain reaction, we revealed differential expression of cellular inhibitor of apoptosis protein-1, inhibitor of kappaB (IkappaB)/IkappaBalpha (MAD-3), and intercellular adhesion molecule-1 (ICAM-1) in SUP(LPS)-stimulated KG-1a cells and up-regulation of interferon (IFN)-inducible T cell-chemoattractant, interleukin (IL)-8, macrophage-inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, RANTES, CD70, granulocyte macrophage-colony stimulating factor, and IL-1beta in stimulated KG-1a cells and CD34(+) BMCs. Although monokine induced by IFN-gamma, IFN-inducible protein 10, and IFN-gamma were exclusively up-regulated in KG-1a cells, differential expression of monocyte chemoattractant protein-1 (MCP-1), macrophage-derived chemokine, myeloid progenitor inhibitory factor-2, and IL-18 receptor was only detectable in CD34(+) BMCs. More importantly, CD34(+) BMCs stimulated by TNF-alpha also showed enhanced secretion of MCP-1, MIP-1alpha, MIP-1beta, and IL-8, and increased ICAM-1 protein expression could be detected in stimulated KG-1a cells and CD34(+) BMCs. Furthermore, we revealed that T cell proliferation can be induced by TNF-alpha-stimulated KG-1a cells, which is preventable by blocking anti-ICAM-1 monoclonal antibodies. Our results demonstrate that CD34(+) HCs have the potential to express a variety of immune-regulatory mediators upon stimulation by inflammatory cytokines including TNF-alpha, which may contribute to innate- and adaptive-immune processes.     

Differential CD4(+) T-cell memory responses induced by two subsets of human monocyte-derived dendritic cells

Dendritic cells (DC) are powerful inducers of primary T-cell responses, but their role in secondary responses has not been extensively analysed. Here, we address the role of two DC subsets derived from human CD16(+) (16(+) mDC) or CD16(-) (16(-) mDC) monocytes on the reactivation of memory responses. CD4(+) CD45RA(-) memory T cells were obtained from adult blood donors, and central (T(CM)) and effector (T(EM)) memory T cells were isolated by fluorescence-activated cell sorting with anti-CCR7 antibodies. The 16(+) mDC and 16(-) mDC were cocultured with autologous lymphocytes, either unpulsed or loaded with purified protein derivatives of Mycobacterium tuberculosis (PPD) or tetanus toxoid (TT), and were analysed for up to 8 days. Over a range of doses, 16(+) mDC drove stronger T-cell proliferative responses against both antigens. Overall, antigen-specific memory cells tended to acquire a phenotype of T(EM) at later time-points in the culture, whereas cells that had completed fewer cycles of division were similar to T(CM). The 16(+) mDC induced higher rates of proliferation on both T(CM) and T(EM) lymphocytes than 16(-) mDC. This phenomenon was not related to the ability of both DC to induce CD25 expression on T cells, to lower secretion of interleukin-2, or to raise production of interleukin-10 during T-cell/16(-) mDC cocultures. The induction of T(CM) effector capacity in terms of interferon-gamma production was faster and more pronounced with 16(+) mDC, whereas both DC had similar abilities with T(EM). In conclusion, these data might reveal new potentials in vaccination protocols with 16(+) mDC aimed at inducing strong responses on central memory T cells.     

Phenotypic and functional comparison of two distinct subsets of programmable cell of monocytic origin (PCMOs)-derived dendritic cells with conventional monocyte-derived dendritic cells

Dendritic cells (DCs) are professional antigen-presenting cells with the ability to induce primary T-cell responses. They are commonly produced by culturing monocytes in the presence of IL-4 and GM-CSF (cells produced in this manner are called conventional DCs). Here we report the generation of two functionally distinct subsets of DCs derived from programmable cells of monocytic origin (PCMOs) in the presence of IL-3 or tumor necrosis factor alpha (TNF-α). Monocytes were treated with macrophage colony-stimulating factor (M-CSF) and IL-3 for 6 days and then incubated with IL-4 and IL-3 (for IL-3 DCs) or with IL-4, GM-CSF and TNF-α (for TNF-α DCs) for 7 days. Monocytes were then loaded with tumor lysate (used as antigen), and poly (I∶C) was added. The maturation factors TNF-α and monocyte conditioned medium (MCM) were added on days 4 and 5, respectively. The phenotypes of the DCs generated were characterized by flow cytometry, and the cells'' phagocytic activities were measured using FITC-conjugated latex bead uptake. T-cell proliferation and cytokine release were assayed using MTT and commercially available ELISA kits, respectively. We found that either IL-3DCs or TNF-α DCs induce T-cell proliferation and cytokine secretion; the cytokine release pattern showed reduced IL-12/IL-10 and IFN-γ/IL-4 ratios in both types of DCs and in DC-primed T-cell supernatant, respectively, which confirmed that the primed T cells were polarized toward aTh2-type immune response. We concluded that PCMOs are a new cell source that can develop into two functionally distinct DCs that both induce a Th2-type response in vitro. This modality can be used as a DC-based immunotherapy for autoimmune diseases.     

Ex vivo expansion does not alter the capacity of umbilical cord blood CD34+ cells to generate functional T lymphocytes and dendritic cells

We examined whether ex vivo expansion of umbilical cord blood progenitor cells affected their capacity to generate immune cells such as T lymphocytes (TLs) and dendritic cells (DCs). The capacity to generate TLs from cord blood CD34(+) cells expanded for 14 days (d14) was compared with that of nonexpanded CD34(+) cells (d0) using fetal thymus organ cultures or transfer into nonobese diabetic/severe combined immunodeficient mice. The cell preparations yielded comparable percentages of immature (CD4(+)CD8(-), CD4(+)CD8(+)) TLs and functional mature (CD3(+)CD4(+), CD3(+)CD8(+)) TLs with an analogous TCR (T-cell receptor)-Vbeta repertoire pattern. As regards DCs, d0 and d14 CD34(+) cells also yielded similar percentages of CD1a(+) DCs with the same expression levels of HLA-DR, costimulatory and adhesion molecules, and chemokine receptors. DCs derived from either d14 or d0 CD34(+) stimulated allogeneic TLs to the same extent, and the cytokine pattern production of these allogeneic TLs was similar with no shift toward a predominant Th1 or Th2 response. Even though the intrinsic capacity of d14 CD34(+) cells to generate DCs was 13-fold lower than that of d0 CD34(+) cells, this reduction was offset by the prior amplification of the CD34(+) cells, resulting in the overall production of 15-fold more DCs. These data indicate that ex vivo expansion of CD34(+) cells does not impair T lymphopoiesis nor DC differentiation capacity.     

Generation and functional characterization of mouse monocyte-derived dendritic cells.

Dendritic cells (DC) are potent antigen-presenting cells with the unique capacity to initiate primary immune responses. As a result, DC are currently used in clinical studies to induce immunity against infectious disease and malignant cells. However, multiple DC subsets exist and it has been suggested that the type of DC may affect the immune response induced. The vast majority of DC used in experimental mouse tumor models is derived from bone marrow progenitors. In contrast, most in vitro as well as in vivo human studies involve the use of DC generated from adherent peripheral blood-derived monocytes in the presence of GM-CSF and IL-4. In the current report, we describe for the first time the generation and characterization of mouse monocyte-derived DC (MODC). The results indicate that mouse MODC display similar morphology, phenotype and immunostimulatory activity as compared to bone marrow-derived DC. Both DC subsets were able to efficiently take up and subsequently cross-present protein antigen to cytotoxic T cells. Moreover, we demonstrate that vaccination with peptide-loaded MODC mediates induction of tumor-reactive immunity in vivo. The isolation and characterization of mouse MODC will provide a valuable research tool to investigate fundamental aspects of DC biology and which DC subsets are most suitable to induce anti-tumor immunity.     

Expression of CD33-related siglecs on human mononuclear phagocytes, monocyte-derived dendritic cells and plasmacytoid dendritic cells

Siglecs are sialic acid binding Ig-like lectins mostly expressed in the haemopoietic and immune systems. Amongst the 11 human siglecs, there are eight proteins highly related to CD33 which have biochemical features of inhibitory receptors, containing two conserved tyrosine-based inhibitory motifs. Five of these (CD33/siglec-3, -5, -7, -9 and -10) are expressed on circulating monocytes. Here we show that monocytes cultured to differentiate into macrophages using either GM-CSF or M-CSF retained expression of these siglecs and their levels were unaffected following stimulation with LPS. In comparison, monocyte-derived dendritic cells down-modulated siglec-7 and -9 following maturation with LPS. Plasmacytoid dendritic cells in human blood expressed siglec-5 only. On monocytes, siglec-5 was shown to mediate rapid uptake of anti-siglec-5 (Fab)2 fragments into early endosomes. This suggests, in addition to inhibitory signalling, a potential role in endocytosis for siglec-5 and the other CD33-related siglecs. Our results show that siglecs are differentially expressed on mononuclear phagocytes and dendritic cells and that some can be modulated by stimuli that promote maturation and differentiation.     

Chromosome analysis after ex vivo expansion of CD34(+) cells from human cord blood

Ex vivo expansion of cord blood hematopoietic progenitors is an attractive way to prepare a sufficient number of transplantable cells for cord blood transplantation in adult patients. The expanded cells need to have genetic stability. Karyotypic analysis of the expanded cells from cord blood CD34(+) cells by 7-day culture with stem cell growth factor, interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor, and erythropoietin was performed. Several-fold increases in total cell number and CFU-GM were 186.7 +/- 62.1 and 27.1 +/- 9.4 (mean +/- standard deviation of data from 6 samples), respectively. Karyotypes were analyzed in 600 expanded cells in total, and all of them showed normal karyotypes. This observation suggests that multifactor supported expansion of cord blood cells may not induce karyotypic abnormality, although a limited number of ex vivo expanded cells were tested.     

Microencapsulated feeder cells as a source of soluble factors for expansion of CD34(+) hematopoietic stem cells

Expansion of hematopoietic stem cells (HSCs) from cord blood is highly desired for treatment and transplantation of adult patients for hematologic diseases. For efficient proliferation of HSCs, CD34(+) cells from cord blood were co-cultured with microencapsulated murine stromal cells (HESS-5) or immortalized human mesenchymal stem cells (MSCs) in their conditioned media (CM). Bioactive substances for HSC proliferation in CM at the onset of culture are likely consumed by HSCs with time, and co-culturing with microencapsulated feeder cells ensures a continuous supply. The cell number of CD34(+) cell progeny efficiently increased under these culture conditions, and progeny were analyzed by flow cytometry, the colony assay and the cobblestone area-forming cell (CAFC) assay. Total nucleated cells and CD34(+) cell number increased 194- and 7.4-fold, respectively, in the presence of microencapsulated HESS-5 in CM. Colony forming cells and CAFCs were well maintained. The effective expansion of total cells and maintenance of primitive progenitor cells suggest that transfusion of the progeny obtained from CD34(+) cell culture with microencapsulated HESS-5 in CM could shorten the time to engraftment by bridging the pancytopenic period and support functional hematopoietic repopulation.     

Gene-expression profiling of CD34+ cells from various hematopoietic stem-cell sources reveals functional differences in stem-cell activity

The replacement of bone marrow (BM) as a conventional source of stem cell (SC) by umbilical cord blood (UCB) and granulocyte-colony stimulating factor-mobilized peripheral blood SC (PBSC) has brought about clinical advantages. However, several studies have demonstrated that UCB CD34(+) cells and PBSC significantly differ from BM CD34(+) cells qualitatively and quantitatively. Here, we quantified the number of SC in purified BM, UCB CD34(+) cells, and CD34(+) PBSC using in vitro and in vivo assays for human hematopoietic SC (HSC) activity. A cobblestone area-forming cell (CAFC) assay showed that UCB CD34(+) cells contained the highest frequency of CAFC(wk6) (3.6- to tenfold higher than BM CD34(+) cells and PBSC, respectively), and the engraftment capacity in vivo by nonobese diabetic/severe combined immunodeficiency repopulation assay was also significantly greater than BM CD34(+), with a higher proportion of CD45(+) cells detected in the recipients at a lower cell dose. To understand the molecular characteristics underlying these functional differences, we performed several DNA microarray experiments using Affymetrix gene chips, containing 12,600 genes. Comparative analysis of gene-expression profiles showed differential expression of 51 genes between BM and UCB CD34(+) SC and 64 genes between BM CD34(+) cells and PBSC. These genes are involved in proliferation, differentiation, apoptosis, and engraftment capacity of SC. Thus, the molecular expression profiles reported here confirmed functional differences observed among the SC sources. Moreover, this report provides new insights to describe the molecular phenotype of CD34(+) HSC and leads to a better understanding of the discrepancy among the SC sources.     

Glucocorticoid amplifies IL-2-dependent expansion of functional FoxP3(+)CD4(+)CD25(+) T regulatory cells in vivo and enhances their capacity to suppress EAE

IL-2 is crucial for the production of CD4(+)CD25(+) T regulatory (Treg) cells while important for the generation of effective T cell-mediated immunity. How to exploit the capacity of IL-2 to expand Treg cells, while restraining activation of T effector (Teff) cells, is an important and unanswered therapeutic question. Dexamethasone (Dex), a synthetic glucocorticoid steroid, has been reported to suppress IL-2-mediated activation of Teff cells and increase the proportion of Treg cells. Thus, we hypothesized that glucocorticoids may be useful as costimulants to amplify IL-2-mediated selective expansion of Treg cells. We show in this study that short-term simultaneous administration of Dex and IL-2 markedly expanded functional suppressive Foxp3(+)CD4(+)CD25(+) T cells in murine peripheral lymphoid tissues. In a myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) mouse model, we observed that splenic CD4(+)CD25(+) T cells failed to suppress the proliferation of CD4(+)CD25(-) T cells. Pretreatment with Dex/IL-2 remarkably increased the proportion of CD4(+)FoxP3(+) cells and partially restored the function of splenic CD4(+)CD25(+) T cells, and inhibited the development of EAE. Therefore, the combination of glucocorticoid and IL-2, two currently used therapeutics, may provide a novel approach for the treatment of autoimmune diseases, transplant rejection and graft-vs.-host disease.     

Interleukin 23 production by intestinal CD103(+)CD11b(+) dendritic cells in response to bacterial flagellin enhances mucosal innate immune defense

Microbial penetration of the intestinal epithelial barrier triggers inflammatory responses that include induction of the bactericidal C-type lectin RegIIIγ. Systemic administration of flagellin, a bacterial protein that stimulates Toll-like receptor 5 (TLR5), induces epithelial expression of RegIIIγ and protects mice from intestinal colonization with antibiotic-resistant bacteria. Flagellin-induced RegIIIγ expression is IL-22 dependent, but how TLR signaling leads to IL-22 expression is incompletely defined. By using conditional depletion of lamina propria dendritic cell (LPDC) subsets, we demonstrated that CD103(+)CD11b(+) LPDCs, but not monocyte-derived CD103(-)CD11b(+) LPDCs, expressed high amounts of IL-23 after bacterial flagellin administration and drove IL-22-dependent RegIIIγ production. Maximal expression of IL-23 subunits IL-23p19 and IL-12p40 occurred within 60 min of exposure to flagellin. IL-23 subsequently induced a burst of IL-22 followed by sustained RegIIIγ expression. Thus, CD103(+)CD11b(+) LPDCs, in addition to promoting long-term tolerance to ingested antigens, also rapidly produce IL-23 in response to detection of flagellin in the lamina propria.     

Implication of delayed TNF-alpha exposure on dendritic cell maturation and expansion from cryopreserved cord blood CD34+ hematopoietic progenitors

Most currently used systems for dendritic cell (DC) production from progenitors entail tumor necrosis factor alpha (TNF-alpha) at the onset of cell culture, based on the notion that TNF-alpha might be required in the early stages of DC development. To optimize conditions for DC expansion from cryopreserved cord blood (CB) CD34+ hematopoietic progenitors, we took a dynamic approach to define the timing of TNF-alpha exposure to the culture. We cultured cord blood CD34+ cells in RPMI-1640 with 10% human AB plasma, stem cell factor (days 1-6), granulocyte-macrophage colony-stimulating factor (days 1-18), interleukin-4 (days 6-18) and varying schedules of TNF-alpha (0-144 h after thawing). Expression of the DC-associated markers, including CD83/CD1a, HLA DR/CD86/CD80, CD14/CD40, was monitored every 3 days. Our data demonstrate that delayed TNF-alpha exposure by 48-72 h after thawing gave rise to two- to three-fold increase in the yield of CD83+ DCs that were highly active in stimulating allogeneic T-cell proliferation compared to immediate TNF-alpha exposure. Thus, the immediate exposure of cryopreserved cord blood CD34+ cells to TNF-alpha, potentially compromising DC expansion, should be avoided. This finding should be of significant consideration when using cryopreserved CD34+ progenitor cells as a source of immunologically competent DCs in a clinical setting.     

Hypoxia modifies proliferation and differentiation of CD34(+) CML cells

We previously showed that hypoxia (1% O(2)) favors the self-renewal of murine and human normal hematopoietic stem cells. This study represents the first attempt to characterize the effects of hypoxia on the maintenance of chronic myeloid leukemia (CML) progenitors. CD34(+) cells isolated from apheresis products of CML patients were incubated in hypoxia (1% O(2)) and normoxia (20% O(2)). After 8 days of culture, their proliferation, capacity for colony-forming-cell (CFC) generation in secondary cultures (pre-CFC), and phenotype (CD34 and platelet-activating factor receptor [PAF-R]) were compared with those of normal cells, and tyrosine phosphorylation in CML cells was measured. Hypoxia inhibits the proliferation of CD34(+) cells and preserves the pre-CFC capacity and cell-surface CD34 expression of CML cells better than normoxia. The PAF-R expression, which was absent on freshly isolated cells, was detected at the cell surface in both populations after 8 days of culture, but with a lower percentage of positive cells in CML cell cultures. Incubation in hypoxia suppressed the PAF-R expression of normal cells and increased it in CML cells, resulting in a similar expression in the two populations. These effects could be linked to inhibition by hypoxia of the tyrosine hyperphosphorylation of cellular proteins, a major hallmark of CML cells.