Interferon gamma selectively inhibits very primitive CD342+CD38- and not more mature CD34+CD38+ human hematopoietic progenitor cells.

To assess the effects of interferon gamma (IFN-gamma) on very primitive hematopoietic progenitor cells, CD34(2+)CD38- human bone marrow cells were isolated and cultured in a two-stage culture system, consisting of a primary liquid culture phase followed by a secondary semisolid colony assay. CD34(2+)CD38- cells needed at least the presence of interleukin 3 (IL-3) and kit ligand (KL) together with either IL-1, IL-6, or granulocyte-colony-stimulating factor (G-CSF) in the primary liquid phase in order to proliferate and differentiate into secondary colony-forming cells (CFC). Addition of IFN-gamma to the primary liquid cultures inhibited cell proliferation and generation of secondary CFC in a dose-dependent way. This was a direct effect since it was also seen in primary single cell cultures of CD34(2+)CD38- cells. The proliferation of more mature CD34+CD38+ cells, however, was not inhibited by IFN-gamma, demonstrating for the first time that IFN-gamma is a specific and direct hematopoietic stem cell inhibitor. IFN-gamma, moreover, preserves the viability of CD34(2+)CD38- cells in the absence of other cytokines. IFN-gamma could, therefore, play a role in the protection of the stem cell compartment from exhaustion in situations of hematopoietic stress and may be useful as stem cell protecting agent against chemotherapy for cancer.

Tumor necrosis factor alpha is a potent synergistic factor for the proliferation of primitive human hematopoietic progenitor cells and induces resistance to transforming growth factor beta but not to interferon gamma.

Since tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and transforming growth factor (TGF)-beta have all been shown to be specific inhibitors of early human hematopoiesis, we wanted to investigate the interactions of these three cytokines on very primitive human adult bone marrow CD34++CD38- hematopoietic progenitor cells, using a pre-colony-forming cell (pre-CFC) assay, which detects the effects of these cytokines on the initial phases of the differentiation of these primitive progenitors, which are unresponsive to interleukin (IL) 3 alone. Surprisingly, TNF-alpha was a very potent stimulator of the proliferation of CD34++CD38- cells and was the most potent synergistic factor for the IL-3-induced proliferation of these cells of all cytokines tested (IL-1, IL-6, granulocyte colony-stimulating factor, kit ligand). TNF-alpha was the only cytokine that, as a single added factor, induced substantial proliferation in CD34++CD38- cells in the presence of IL-3, except for kit ligand, which induced very limited proliferation. TNF-alpha, moreover, induced a high degree of resistance to the inhibitory effects of TGF-beta in a dose-dependent way. The inhibitory effects of IFN-gamma, however, were not affected by the presence of TNF-alpha. We hypothesize that in situations of the hematopoietic stress, TNF-alpha may abrogate the inhibitory effect of ambient TGF-beta in the bone marrow microenvironment to allow primitive stem cells to proliferate and differentiate in response to an increased demand for mature blood cells.

Proinflammatory response of human leukemic cells to dsRNA transfection linked to activation of dendritic cells.

Leukemic cells exert immunosuppressive effects that interfere with dendritic cell (DC) function and hamper effective antileukemic immune responses. Here, we sought to enhance the immunogenicity of leukemic cells by loading them with the double-stranded (ds) RNA Toll-like receptor 3 (TLR3) ligand polyriboinosinic polyribocytidylic acid (poly(I:C)), mimicking viral infection of the tumor cells. Given the responsiveness of DC to TLR ligands, we hypothesized that the uptake of poly(I:C)-loaded leukemic cells by immature DC (iDC) would lead to DC activation. Primary acute myeloid leukemia (AML) cells and AML cell lines markedly responded to poly(I:C) electroporation by apoptosis, upregulation of TLR3 expression, enhanced expression of major histocompatibility complex (MHC) and costimulatory molecules and by production of type I interferons (IFN). Upon phagocytosis of poly(I:C)-electroporated AML cells, DC maturation and activation were induced as judged by an increased expression of MHC and costimulatory molecules, production of proinflammatory cytokines and an increase of T helper 1 (T(H)1)-polarizing capacity. These immune effects were suboptimal when AML cells were passively pulsed with poly(I:C), indicating the superiority of poly(I:C) transfection over pulsing. Our results demonstrate that poly(I:C) electroporation is a promising strategy to increase the immunogenicity of AML cells and to convert iDC into activated mature DC following the phagocytosis of AML cells.

Flow cytometric cell cycle analysis allows for rapid screening of estrogenicity in MCF-7 breast cancer cells.

The quantitative measurement of individual cells and their characteristics by means of flow cytometry is already for many years of great value for clinical studies. However, its potential as a tool in (eco)toxicology has only recently been discovered. Analysis of cell cycle kinetics with DNA-staining dyes can offer a valuable alternative to detect effects of chemicals on cell proliferation, an important endpoint in screening estrogen-like properties of chemicals. In the present study, flow cytometric cell cycle analysis in growth arrested MCF-7 cells exposed to five xenoestrogens correspond well with cell proliferation results of the conventionally used E-screen assay. Moreover, re-induction of proliferation in MCF-7 cells, indicated by the percentage of cells in S(ynthesis)-phase, is most pronounced after 24 h exposure, thus allowing a faster screening of xenoestrogens. This flow cytometric proliferation assay confirms that the estrogenic activity of structurally analogous parabens is mediated by the estrogen receptor pathway and is proportional to the alkyl chain length. Moreover, the ER-mediated mode of action of two fluorotelomer alcohols (6:2 FTOH and 8:2 FTOH), recently reported as xenoestrogenic, could be elucidated. These results support the potential of flow cytometric cell cycle kinetics as a screening assay for estrogen-like properties of chemicals.

RNA-based gene transfer for adult stem cells and T cells.

Electroporation of mRNA has become an established method for gene transfer into dendritic cells for immunotherapeutic purposes. However, many more cell types and applications might benefit from an efficient mRNA-based gene transfer method. In this study, we investigated the potential of mRNA-based gene transfer to induce short-term transgene expression in adult stem cells and activated T cells, based on electroporation with mRNA encoding the enhanced green fluorescent protein. The results show efficient transgene expression in CD34-positive hematopoietic progenitor cells (35%), in in vitro cultured mesenchymal cells (90%) and in PHA-stimulated T cells (50%). Next to presentation of gene transfer results, potential applications of mRNA-based gene transfer in stem cells and T cells are discussed.

The combined effects of all-trans retinoic acid and TGF-beta on the initial proliferation of normal human bone marrow progenitor cells.

We investigated the cell kinetic effects of retinoic acid (RA) and the functional interaction between RA and TGF-beta on normal human bone marrow progenitor cells (CD34+). Cell cycle progression throughout the first three consecutive cell cycles and alterations in cell kinetic responses were measured using the BrdU-Hoechst quenching technique. RA stimulates the IL-3-induced growth by additionally recruiting quiescent stem and progenitor cells out of the G0/G1-phase and by increasing the cell cycle traverse rate. In contrast, TGF-beta addition resulted in a significant decrease in the number of proliferating cells. Simultaneous addition of RA and TGF-beta resulted in a stronger inhibition compared to addition of TGF-beta alone. Preincubation experiments further showed that RA is capable of sensitizing the progenitors to the inhibitory action of TGF-beta: the inhibitory effect of TGF-beta was significantly increased when cells were pretreated with RA. These data show that, in combination with IL-3, RA additionally stimulates quiescent bone marrow progenitors in a simultaneous way, and that it increases sensitivity of the progenitors to the inhibitory action of TGF-beta. The combination of RA and TGF-beta on normal and leukemic hematopoiesis has to be further investigated, since this combination may possibly provide additional therapeutic benefit.

Interleukin 4 and interferon gamma costimulate the expansion of early human myeloid colony-forming cells. Proposal of a model for the regulation of myelopoiesis by interleukin 4 and interferon gamma and its integration with the regulation of the immune response.

We have previously shown that interleukin 4 (IL-4) and interferon gamma (INF-gamma) reciprocally regulate the production of granulocytes and monocytes from mature monopotential hematopoietic progenitor cells, while at the level of the very primitive stem cells IFN-gamma is a selective inhibitor of proliferation and differentiation, and IL-4 has weak stimulatory effects. We investigated the effects of IL-4 and IFN-gamma on the expansion in suspension culture of myeloid colony-forming cells (CFCs) induced by either IL-3 or IL-1+IL-3, using on the one hand more differentiated CD34+HLA-DR strongly positive (HLA-DR++) and on the other hand more primitive Cd34+HLA-DR weakly positive (HLA-DR+/-) human bone marrow cells. It is shown that both IL-4 and IFN-gamma stimulate the IL-3- and IL-3+IL-1-induced expansion of the number of CFCs in the HLA-DR+/- population. In the presence, but not in the absence of IL-1, additive effects of IL-4 and IFN-gamma were seen. We could not demonstrate any IL-3-like effect by IL-4 on early human hematopoietic progenitors. No expansion of CFC number was seen in the HLA-DR++ population. Based on these data and on data which we have published previously, a model for the regulation of myelopoiesis by IL-4 and IFN-gamma is proposed. In this model, IL-4 and IFN-gamma, which are both immune recognition induced inflammatory cytokines, both stimulate the expansion and recruitment of early myeloid progenitors, whereas at the level of their terminal differentiation, the balance between both cytokines determines whether preferentially monocytes/macrophages (IFN-gamma) or granulocytes (IL-4) are being produced. At the level of the most primitive cells, the inhibitory action of IFN-gamma might prevent differentiative exhaustion of the stem cell compartment in situations of hematopoietic stress.

mRNA-electroporated mature dendritic cells retain transgene expression, phenotypical properties and stimulatory capacity after cryopreservation.

Genetically modified dendritic cells (DC) are increasingly used in vitro to activate cytotoxic T lymphocyte (CTL) immune responses. Because T cell activation protocols consist of multiple restimulation cycles of peripheral blood lymphocytes with antigen-loaded mature DC, continuous generation of DC is needed throughout the experiment. Therefore, cryopreservation of DC loaded with antigen is a valuable alternative for weekly generation and modification of DC. Recently, we described an antigen loading method for DC based on electroporation of defined tumor antigen mRNA. In this study, we demonstrate that mRNA-electroporated DC can efficiently be prepared for cryopreservation. Using an optimized maturation and freezing protocol after mRNA electroporation, we obtained high transgene-expressing viable mature DC. In addition, we showed that these modified cryopreserved DC retain stimulatory capacity in an influenza model system. Therefore, cryopreservation of mature mRNA-electroporated DC is a useful method for continuous availability of antigen-loaded DC throughout T cell activation experiments.

Antiproliferative effect of plant cytokinin analogues with an inhibitory activity on cyclin-dependent kinases.

In this study, analogues of olomoucine, a previously described plant cytokinin analogue with cyclin-dependent kinase (CDK) inhibitory activity, were investigated for effect on CDK1 and CDK2 and for effect on cell proliferation. Eight new compounds exhibit stronger inhibitory activity on CDK1 and CDK2 and on cell proliferation than olomoucine. Some active compounds showed low inhibition of proliferation of normal myeloid growth. Improvement of inhibitory activity of known compounds with a C6-benzylamino group was brought about by substitution with one hydroxyl. Also, new C2 substituents associated with inhibitory activity on CDK and on cell proliferation are described. There was a significant correlation between effect on CDK and antiproliferative effect on the KG1 and Molt3 cell lines and on primary human lymphocytes, strongly suggesting that at least part of the antiproliferative effect of cytokinin analogues was due to inhibition of CDK activity. Cytokinin analogues induced apoptosis in a time- and concentration-dependent manner and changes in cell cycle distribution. The antiproliferative and pro-apoptotic effects of plant cytokinin analogues suggest that they are a new class of cytostatic agents and that they may find an application in the chemotherapy of cancer.

Developmentally regulated responsiveness to transforming growth factor-beta is correlated with functional differences between human adult and fetal primitive hematopoietic progenitor cells.

Important functional differences exist between primitive CD34++ CD38- hematopoietic progenitor cells derived from human fetal liver (FL) and adult bone marrow (ABM). FL progenitors are known to have higher proliferative capacities and lower cytokine requirements than their ABM counterparts. In this study, we isolated FL and ABM CD34++ CD38- cells and used a two-stage culture system to investigate the effects of transforming growth factor-beta (TGF-beta) and blocking anti-TGF-beta antibodies (anti-TGF-beta) on these cells. First, we demonstrate that FL progenitors are significantly less sensitive to the inhibitory effects of TGF-beta than ABM cells. Second, whereas ABM cells are significantly stimulated by anti-TGF-beta, only very limited effects are seen on FL cells. Third, we show that the effect of anti-TGF-beta is mainly situated at the level of the initial cell cycles of very primitive progenitor cells with a high proliferation potential. Fourth, we demonstrate that blocking the effects of endogenous TGF-beta reduces the growth factor requirements of ABM cells in order to proliferate and differentiate. Based on these data, we hypothesize that at least part of the functional differences that exist between adult and fetal stem cells can be accounted for by a developmental different responsiveness to TGF-beta.

Differential regulation of the expression of CD38 and human leukocyte antigen-DR on CD34+ hematopoietic progenitor cells by interleukin-4 and interferon-gamma.

We studied the effects of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) on the expression of CD38 and human leukocyte antigen (HLA)-DR on purified CD34+ bone marrow progenitor cells. CD34+CD38- and CD34+HLA-DR- cells are largely nonoverlapping populations. After culture for 4 days in IFN-gamma, the expression of CD38 and HLA-DR is significantly increased and the disappearance of the CD38- and HLA-DR- populations is virtually complete. Moreover, IFN-gamma induces a population of CD34+ cells with a very high expression of CD38 (CD34+CD38++ cells), which were absent in the initial CD34+ population. IL-4 has no effect on the expression of CD38, but induces a limited but significant increase in the expression of HLA-Dr. After culture in IFN-gamma, CD34+ cells show a higher cloning efficiency of the colony-forming unit-macrophage (CFU-M) and burst-forming unit-erythroid (BFU-E) compared to cells cultured in medium alone. After culture in IL-4, a limited increase in CFU-granulocyte (CFU-G) and BFU-E is seen, whereas CFU-G, CFU-M, and BFU-E are increased after culture in IL-4 plus IFN-gamma. We further investigated the functional properties of the CD34+CD38++ cells generated in the presence of IFN-gamma. This cell population is highly enriched for BFU-E but partially depleted of CFU-M. Most of the CFU-M were found in the CD34+CD38+/-(CD34+CD38- and CD34+CD38+ cells) population.

Transforming growth factor-beta regulates the cell cycle status of interleukin-3 (IL-3) plus IL-1, stem cell factor, or IL-6 stimulated CD34+ human hematopoietic progenitor cells through different cell kinetic mechanisms depending on the applied stimulus.

The immediate cell kinetic response of highly purified human bone marrow progenitor cells (CD34+ sorted fraction) to the inhibitory effects of transforming growth factor-beta (TGF-beta) was studied using the BrdU-Hoechst flow-cytometric technique. The progenitor cells were stimulated with either interleukin-3 (IL-3) alone or with IL-3 in combination with IL-1, stem cell factor (SCF), or IL-6, and the inhibitory action of TGF-beta was evaluated in each phase of the first three consecutive cell cycles. Semisolid methylcellulose cultures were also performed to compare these initial events to the effects observed after 7, 14, and 21 days of incubation. Within the CD34+ compartment, the progenitor cells can be discriminated on a functional basis, i.e., in terms of TGF-beta sensitivity. Very primitive progenitors, recruited out of the G0 phase by IL-3 plus an early-acting factor (IL-1, SCF) are, upon addition of TGF-beta, arrested specifically in the G1 phase of the second cell cycle. In the clonogenic assays, the increased colony formation due to IL-1 or SCF was completely abolished by the counteracting effect of TGF-beta that diminished colony output back to the level of TGF-beta-plus-IL-3 supplemented colony growth. Addition of TGF-beta to CD34+ progenitors responding to IL-3 alone resulted in an overall retardation, but without an apparent specific accumulation of cells in any of the cell cycles. Finally, within the CD34+ compartment, there exists a subset of IL-3-responsive, but TGF-beta-insensitive, progenitor cells that were, upon addition of TGF-beta, not arrested at all. In conclusion, our results demonstrate that TGF-beta exerts different cell kinetic effects on CD34+ progenitor cell growth depending on the applied stimulus.

TGF-beta and MIP-1 alpha exert their main inhibitory activity on very primitive CD34+2CD38- cells but show opposite effects on more mature CD34+CD38+ human hematopoietic progenitors.

We investigated the effects of transforming growth factor-beta (TGF-beta) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) on very primitive CD34++CD38- and on more mature CD34++CD38+ human hematopoietic progenitor cells by means of a two stage pre-colony-forming cell (pre-CFC) assay. The first (liquid) stage of this assay allows evaluation of the effects of TGF-beta and MIP-1 alpha on the "primary" proliferation of the progenitors under study and on the generation of "secondary" colony-forming cells (CFC, cells for which a second stage semisolid clonogenic assay was used as a read-out). TGF-beta inhibited the proliferation and CFC generation of CD34++CD38- and CD34+CD38+ cells, showing the strongest inhibitory activity on CD34++CD38- cells. MIP-1 alpha exerted a weaker inhibitory activity on CD34+2CD38- cells, whereas it enhanced the primary proliferation of CD34+CD38+ cells and generation of secondary CFC in this subpopulation. Thus, TGF-beta, and MIP-1 alpha both inhibit very primitive CD34+2)CD38- cells, but they are not equally potent. The effects of TGF-beta and MIP-1 alpha on more mature progenitor cells are more complex. Our results and data from the literature indicate that, as progenitor cells mature, they reach a "pivotal point" at a certain stage in their differentiation pathway, depending on the inhibitor, where they are no longer inhibited or where they may even be stimulated by the former inhibitor to proliferate.

CD34++ CD38- and CD34+ CD38+ human hematopoietic progenitors from fetal liver, cord blood, and adult bone marrow respond differently to hematopoietic cytokines depending on the ontogenic source.

CD34++ CD38- and CD34+ CD38+ hematopoietic progenitor cells (HPCs) from human fetal liver (FL), cord blood (CB), and adult bone marrow (ABM) were isolated and investigated for their growth characteristics, cytokine requirements and response to two modulators of early hematopoiesis, interferon (IFN)-gamma and macrophage inflammatory protein (MIP)-1alpha. We observed first that a significantly lower percentage of CD34++ cells were CD38- in ABM than in FL and CB. Second, the functional differences between CD34++ CD38- and CD34+ CD38+ cells were less pronounced in FL and CB than in their ABM counterparts. Third, an inverse correlation was found between growth factor response and the ontogenic age of HPCs, and a direct correlation was noted between cytokine requirements and the ontogenic age of HPCs. Fourth, spontaneous colony formation in a classic semisolid culture system was reproducibly obtained only in the ontogenically earliest cells, that is, in FL but not in CB and ABM, in which no such spontaneous colony formation was observed. Fifth, the modulatory effects of IFN-gamma and MIP-1alpha were qualitatively different depending on the ontogenic age of the progenitor source: whereas IFN-gamma was only a selective inhibitor of primitive CD34++ CD38- ABM progenitor cells, it inhibited both CD34++ CD38- and CD34+ CD38+ FL and CB cells to the same extent. In contrast to the effects of MIP-1alpha on ABM, we could not find any consistently stimulatory or inhibitory effects on FL and CB progenitors. In conclusion, important functional and biologic differences exist between FL, CB, and ABM progenitor cells, and these differences could have major implications for the use of these cell populations in preparative protocols of ex vivo expansion, transplantation strategies, or gene transfer experiments.

A low content in zeatin type cytokinins is not restrictive for the occurrence of G1/S transition in tobacco BY-2 cells.

Theories on the importance of cytokinins in G1/S transition control are manifold and contradictory. By establishing a double A(phi-PZ block, maximal synchronization of a BY-2 suspension culture was obtained to investigate the effect of cytokinin depletion on G1/S transition. Lovastatin was used as a specific inhibitor of cytokinin biosynthesis. Flow cytometry showed that the G1/S transition occurred regardless of the cytokinin drop. This observation indicates an extremely low dose requiry for that stage of the cell cycle. It is very likely that precisely the downregulation of zeatin type cytokinins matters for the G1/S transition to occur, since cytokinin addition at early G1 blocked the cycle at G1/S.

In vitro effect of ketone bodies, glucocorticosteroids and bovine pregnancy-associated glycoprotein on cultures of bone marrow progenitor cells of cows and calves.

Changes in the number, maturity and function of neutrophils, concomitant changes in plasma concentrations of hormones and metabolites, and the increased susceptibility of cows to infectious diseases around parturition, led us to investigate the effect of beta-hydroxybutyric acid (BHBA), acetoacetic acid (AcAc), hydrocortisone-21-acetate (HCAc) and bovine pregnancy-associated glycoprotein (bPAG) on the proliferation of bovine bone marrow progenitor cells in methylcellulose in vitro cultures. Myeloid progenitors were stimulated with concanavalin A-stimulated leukocyte conditioned medium (LCM) and erythroid progenitors with erythropoietin in the presence of hemin. Erythroid and myeloid colonies were scored after five and seven days, respectively. BHBA and AcAc induced inhibitory effects on the proliferation of bovine bone marrow cells at concentrations of 1.0, 2.5, and 5.0 mM. HCAc significantly inhibited growth of progenitors at concentrations of 10, 20, 50, and 100 ng/ml, and bPAG at concentrations of 2400 and 3000 ng/ml. The results of this study suggest that in the cow high concentrations of BHBA, AcAc, HCAc and bPAG, which can be reached in the circulation around calving, could alter the number of circulating neutrophils after parturition. This phenomenon might contribute to the increased susceptibility of dairy cows to environmental mastitis.

Culture of bovine bone marrow progenitor cells in vitro.

In vitro methylcellulose cultures of bovine bone marrow progenitor cells were developed. An existing technique described for bovine species was compared to a method for human tissue and further adapted during subsequent experiments. Bovine bone marrow samples were collected at the slaughterhouse, and mononuclear cells were separated by gradient centrifugation (1.077 g/ml specific density and 400 g). The use of 3% bovine leucocyte-conditioned medium, produced by stimulation of blood lymphocytes with 4 microg/ml concanavalin A and harvested on day 4 of culture, gave better results than the use of supernatant of the human bladder carcinoma 5637, which is widely used in human bone marrow cultures. However, bovine leucocyte-conditioned medium was not added to erythroid cultures because inhibitory effects were observed. Erythroid colonies were stimulated with erythropoietin, and hemin was added to enable microscopic identification. Reduced oxygen tension was necessary to induce growth of erythroid colonies. This was not necessary for myeloid cultures. In conclusion, the results of this study show that the growth of myeloid and erythroid colonies in methylcellulose-based medium requires different culture conditions, which are different from the culture conditions for human cells.

Flow cytometric analysis of the DNA content of bovine and human bone marrow cells.

The defence against infection in high-yielding dairy cows is correlated with the number and function of circulating neutrophils and depends on their production in bone marrow. Therefore, the DNA content of isolated bone marrow cell suspensions from 7 calves, 7 cows and 14 humans was assayed by flow cytometry. Bovine sternal bone marrow samples were collected within 30 min of death, and human marrow samples were collected by sternal puncture and aspiration. Mononucleated cells were isolated by gradient centrifugation. In the bone marrow samples from calves and cows, 35 +/- 2.6% and 31.8 +/- 1.5% of the isolated bone marrow cells respectively were in the S/G2/M-phase. The difference between calves and cows was not significant. In the human samples, only 12 +/- 0.8% of the cells were in the S/G2/M-phase. A significant (P < 0.001) difference was observed between the two species. These results indicated that the proliferative, in activity of haematopoietic cells is significantly higher in cattle than in humans.

Immunosuppression induced by immature dendritic cells is mediated by TGF-beta/IL-10 double-positive CD4+ regulatory T cells.

Dendritic cells (DC) have important functions in T cell immunity and T cell tolerance. Previously, it was believed that T cell unresponsiveness induced by immature DC (iDC) is caused by the absence of inflammatory signals in steady-state in vivo conditions and by the low expression levels of costimulatory molecules on iDC. However, a growing body of evidence now indicates that iDC can also actively maintain peripheral T cell tolerance by the induction and/or stimulation of regulatory T cell populations. In this study, we investigated the in vitro T cell stimulatory capacity of iDC and mature DC (mDC) and found that both DC types induced a significant increase in the number of transforming growth factor (TGF)-beta and interleukin (IL)-10 double-positive CD4(+) T cells within 1 week of autologous DC/T cell co-cultures. In iDC/T cell cultures, where antigen-specific T cell priming was significantly reduced as compared to mDC/T cell cultures, we demonstrated that the tolerogenic effect of iDC was mediated by soluble TGF-beta and IL-10 secreted by CD4(+)CD25(-)FOXP3(-) T cells. In addition, the suppressive capacity of CD4(+) T cells conditioned by iDC was transferable to already primed antigen-specific CD8(+) T cell cultures. In contrast, addition of CD4(+) T cells conditioned by mDC to primed antigen-specific CD8(+) T cells resulted in enhanced CD8(+) T cell responses, notwithstanding the presence of TGF-beta(+)/IL-10(+) T cells in the transferred fraction. In summary, we hypothesize that DC have an active role in inducing immunosuppressive cytokine-secreting regulatory T cells. We show that iDC-conditioned CD4(+) T cells are globally immunosuppressive, while mDC induce globally immunostimulatory CD4(+) T cells. Furthermore, TGF-beta(+)/IL-10(+) T cells are expanded by DC independent of their maturation status, but their suppressive function is dependent on immaturity of DC.