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.

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.

Ex vivo induction of viral antigen-specific CD8 T cell responses using mRNA-electroporated CD40-activated B cells.

Cell-based immunotherapy, in which antigen-loaded antigen-presenting cells (APC) are used to elicit T cell responses, has become part of the search for alternative cancer and infectious disease treatments. Here, we report on the feasibility of using mRNA-electroporated CD40-activated B cells (CD40-B cells) as alternative APC for the ex vivo induction of antigen-specific CD8(+) T cell responses. The potential of CD40-B cells as APC is reflected in their phenotypic analysis, showing a polyclonal, strongly activated B cell population with high expression of MHC and co-stimulatory molecules. Flow cytometric analysis of EGFP expression 24 h after EGFP mRNA-electroporation showed that CD40-B cells can be RNA transfected with high gene transfer efficiency. No difference in transfection efficiency or postelectroporation viability was observed between CD40-B cells and monocyte-derived dendritic cells (DC). Our first series of experiments show clearly that peptide-pulsed CD40-B cells are able to (re)activate both CD8+ and CD4(+) T cells against influenza and cytomegalovirus (CMV) antigens. To demonstrate the ability of viral antigen mRNA-electroporated CD40-B cells to induce virus-specific CD8+ T cell responses, these antigen-loaded cells were co-cultured in vitro with autologous peripheral blood mononuclear cells (PBMC) for 7 days followed by analysis of T cell antigen-specificity. These experiments show that CD40-B cells electroporated with influenza M1 mRNA or with CMV pp65 mRNA are able to activate antigen-specific interferon (IFN)-gamma-producing CD8(+) T cells. These findings demonstrate that mRNA-electroporated CD40-B cells can be used as alternative APC for the induction of antigen-specific (memory) CD8(+) T cell responses, which might overcome some of the drawbacks inherent to DC immunotherapy protocols.

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.

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.

Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells.

Designing effective strategies to load human dendritic cells (DCs) with tumor antigens is a challenging approach for DC-based tumor vaccines. Here, a cytoplasmic expression system based on mRNA electroporation to efficiently introduce tumor antigens into DCs is described. Preliminary experiments in K562 cells using an enhanced green fluorescent protein (EGFP) reporter gene revealed that mRNA electroporation as compared with plasmid DNA electroporation showed a markedly improved transfection efficiency (89% versus 40% EGFP(+) cells, respectively) and induced a strikingly lower cell toxicity (15% death rate with mRNA versus 51% with plasmid DNA). Next, mRNA electroporation was applied for nonviral transfection of different types of human DCs, including monocyte-derived DCs (Mo-DCs), CD34(+) progenitor-derived DCs (34-DCs) and Langerhans cells (34-LCs). High-level transgene expression by mRNA electroporation was obtained in more than 50% of all DC types. mRNA-electroporated DCs retained their phenotype and maturational potential. Importantly, DCs electroporated with mRNA-encoding Melan-A strongly activated a Melan-A-specific cytotoxic T lymphocyte (CTL) clone in an HLA-restricted manner and were superior to mRNA-lipofected or -pulsed DCs. Optimal stimulation of the CTL occurred when Mo-DCs underwent maturation following mRNA transfection. Strikingly, a nonspecific stimulation of CTL was observed when DCs were transfected with plasmid DNA. The data clearly demonstrate that Mo-DCs electroporated with mRNA efficiently present functional antigenic peptides to cytotoxic T cells. Therefore, electroporation of mRNA-encoding tumor antigens is a powerful technique to charge human dendritic cells with tumor antigens and could serve applications in future DC-based tumor vaccines.

Establishment of serum-free pre-colony forming unit assays for differentiation of primitive hematopoietic progenitors: serum induces early macrophage differentiation and inhibits early erythroid differentiation of CD34++CD38- cells.

In this report we show that serum has differentiation-inducing effects on primitive hematopoietic progenitor cells with the CD34++CD38- immunophenotype. Using the pre-colony forming unit (pre-CFU) assay as a model for early myelopoiesis, we compared the effects of serum-containing and serum-free media and evaluated different cytokine cocktails [interleukin (IL)-1, IL-3, IL-6, kit ligand with and without the Flt3/Flk2 ligand (FL)]. In this assay, pre-CFUs are defined as cells unable to form colonies when plated directly in semi-solid assays, but which can differentiate into CFUs when cultured in liquid medium containing early-acting cytokines. In one of the investigated serum-free media, the average myeloid expansion in liquid medium reached up to more than 50% of that obtained in serum-containing medium. In addition, our experiments revealed differences in the clonogenic output between cells cultured in serum-free medium and those cultured in serum-containing medium, demonstrating that serum has a monocyte differentiation-inducing effect on primitive hematopoietic progenitors. Also in serum-free medium, higher proportions of erythroid progenitors were generated. These differentiation-inducing effects of serum further emphasize the need for serum-free culture protocols for hematopoietic graft engineering. Addition of FL to the culture media ameliorated cellular expansion and resulted in a decrease in the proportion of erythroid and granulocyte progenitors and an increase in the proportion of monocyte progenitors. In conclusion, this study shows that good serum-free conditions are available for differentiation assays with primitive hematopoietic progenitors and demonstrates that serum and FL have biasing effects on the initial phase of hematopoietic differentiation, favoring the monocyte lineage.

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.

High-level transgene expression in primary human T lymphocytes and adult bone marrow CD34+ cells via electroporation-mediated gene delivery.

The design of effective gene delivery systems for gene transfer in primary human blood cells is important both for fundamental hematopoiesis research and for cancer gene therapy strategies. Here, we evaluated electroporation as a nonviral means for transfection of activated human T lymphocytes and adult bone marrow (BM) CD34+ cells. We describe optimal culture and electroporation parameters for efficient gene delivery in prestimulated T lymphocytes (16.3 +/-1.3%), as well as 2-day cultured adult BM CD34+ cells (29.6+/-4.6%). PHA-stimulated T cells were most receptive for transfection after 48h of in vitro culture, while T cells stimulated by CD3 cross-linking and interleukin (IL)-2 achieved maximum transfection levels after 72 h of prestimulation. Kinetic analysis of EGFP expression revealed that activated T lymphocytes maintained transgene expression at high levels for a prolonged period. In addition, fresh unstimulated BM CD34+ cells were consistently transfected (5.2+/-0.4%) with minimal cytotoxicity (<5%), even without preliminary CD34+ cell purification. Both T cells and CD34+ cells retained their phenotype and functional capacity after electroporation. These results demonstrate that electroporation is a suitable nonviral transfection technique that may serve applications in gene therapy protocols using T lymphocytes or CD34+ 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.

Generation of T cells from adult human hematopoietic stem cells and progenitors in a fetal thymic organ culture system: stimulation by tumor necrosis factor-alpha.

To investigate the T-lymphopoietic capacity of human adult bone marrow (ABM) hematopoietic progenitor cells, CD34+Lin-, CD34+CD38+, and CD34++CD38- cells were cultured in a severe combined immunodeficient (SCID) mouse fetal thymic organ culture (FTOC). Direct seeding of these progenitors resulted in a moderate to severe cell loss, particularly for the CD34++CD38- cell fraction, and T cells could only be generated from the CD34+Lin- fraction. Preincubation for 36 hours with interleukin-3 (IL-3) and stem cell factor (SCF) led to an improved cell survival and proliferation, although T-cell development was seen only in the CD34+Lin- fraction. Addition of tumor necrosis factor (TNF)-alpha to IL-3 + SCF-supplemented preincubation medium resulted in optimal cell survival, cell proliferation. and T-cell generation of all 3 cell fractions. The TNF-alpha effect resulted in an up-regulation of CD127 (ie, the IL-7 receptor alpha-chain) in a small subset of the CD34+ cells. No evidence could be generated to support the possibility that TNF-alpha inhibits a cell population that suppresses T-cell differentiation. A quantitatively different T-cell generation potency was still seen between the 3 subpopulations: CD34+Lin- (100% success rate) > CD34+CD38+ (66%) > CD34++CD38- (25%). These data contrast with our previous findings using fetal liver and cord blood progenitors, which readily differentiate into T-lymphocytes in FTOC, even without prestimulation with cytokines. Our results demonstrate that adult CD34++CD38- cells, known to contain hematopoietic stem cells, can differentiate into T-lymphocytes and that a significant difference exists in T-lymphopoietic activity of stem cells derived from ontogenetically different sources. (Blood. 2000;95:2806-2812)

GP130 and c-kit signalling, initiated by the sIL-6R/IL-6 complex, is insufficient to expand the primitive adult bone marrow CD34+CD38- pre-CFU cell.

It has previously been shown that gp130 and c-kit signalling synergize for the ex vivo expansion of human cord blood (CB) CD34+ haematopoietic progenitor cells. We were interested in evaluating this synergy within an ontogenetically different haematopoietic tissue [i.e. adult bone marrow (BM)] and on a more primitive progenitor subset (i.e. CD34+ CD38-cells), which are highly enriched for pre-colony forming unit (CFU) cells. These cells were plated out in a primary liquid culture supplemented with either interleukin (IL)-6+stem cell factor (SCF), IL-6+ SCF+soluble IL-6 receptor (sIL-6R), IL-6+SCF+sIL-6R+IL3+IL-1 or SCF+IL-3+IL-6+IL-1. Cell counting after liquid culture revealed an absolute expansion of 2.2-, 4.1-, 89.5- and 65.7-fold compared with initial cell input for the four-cytokine combinations, respectively. The secondary read-out assay revealed that this cell expansion in the liquid culture also resulted in CFU generation, with absolute cloning efficiencies of 0.002, 0.024, 12.13 and 7.73 (per cell initially present) for the respective cytokine combinations. These results indicate that gp130 and c-kit signalling alone (i.e. using IL6+SCF+sIL-6R), in terms of both cell number and CFU generation, insufficiently stimulate primitive adult BM CD34+CD38- haematopoietic cells in order to reach a CFU generation comparable with that obtained after multifactor stimulation. Adding sIL-6R to the multifactor stimulation and compared with this multifactor stimulation, a 1.7-fold synergy in terms of cell expansion and a 3.0-fold synergy in terms of CFU generation are obtained. The sIL-6R/IL-6 complex thus has a narrower spectrum of action on primitive adult BM CD34+CD38- cells than on CB CD34+ cells.

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.

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.

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.

Nonviral transfection of distinct types of human dendritic cells: high-efficiency gene transfer by electroporation into hematopoietic progenitor- but not monocyte-derived dendritic cells.

Human dendritic cells (DC) are highly professional antigen presenting cells for the priming of naive cytotoxic T cells. Gene transfer in DC would be a useful strategy to load DC with relevant de novo synthesized antigens for immunotherapeutical purposes. As a first step towards a DC-based gene therapy, we examined the efficiency of nonviral transfection in different types of cultured human dendritic cells with a humanized red-shifted green fluorescent protein reporter gene. Plasmid DNA transfection by electroporation or lipofection was used to transfect CD34+ progenitor cell-derived DC (PC-DC) and Langerhans' cells (PC-LC), as well as monocyte-derived DC (Mo-DC). While lipofection was unsuccessful in all types of DC, we obtained high-efficiency gene transfer by electroporation in PC-LC (16%) and PC-DC (12%). In contrast, electroporation was strikingly less efficient in Mo-DC (< or = 2%). The potent allostimulatory capacity of DC was still retained in electroporated PC-DC and PC-LC. In conclusion, electroporation of antigen expressing plasmid DNA is an efficient tool for nonviral gene transfer in PC-DC and PC-LC, but not in Mo-DC and could be useful for the development of DC-based tumor immunotherapy.

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.

Decrease in nucleoside diphosphate kinase (NDPK/nm23) expression during hematopoietic maturation.

The nucleoside diphosphate kinase (NDPK/nm23) isoforms H1 and H2 were localized in hematopoietic tissues. Flow cytometric analysis and enzymatic assays were used to quantify the intracellular and extracellular concentrations of NDPK. Bone marrow CD34(+) progenitors contained the highest intracellular levels of both nm23-H1 and nm23-H2. Lower levels were measured in more mature bone marrow cells, whereas peripheral blood leukocytes had the lowest expression of nm23. These data suggest a function of NDPK in early hematopoiesis and a down-regulation of NDPK upon differentiation. In addition, an up-regulation of nm23 expression was observed in lymphocytes after induction of proliferation with phytohemagglutinin. Multiparameter flow cytometry demonstrated that this up-regulation occurred during the G0/G1-transition. Flow cytometric analysis also revealed a weak surface expression of nm23 on a number of hematopoietic cell lines, which was not detected on normal hematopoietic cells. Our data also demonstrated the presence of NDPK in human plasma, probably due to a limited in vivo lysis of red blood cells.