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.

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.

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.

Antigen-specific cellular immunotherapy of leukemia.

Advances in cellular and molecular immunology have led to the characterization of leukemia-specific T-cell antigens and to the development of strategies for effective augmentation of T-cell immunity in leukemia patients. While several leukemia-related antigens have been identified, this review focuses on the Wilms' tumor 1 (WT1) antigen and the proteinase 3 (Pr3) antigen that are overexpressed in leukemic cells and are already being used in the clinical setting. Moreover, WT1 is also overexpressed in a vast number of nonhematological solid tumors, thereby expanding its use as a promising target for cancer vaccines. Examples of spontaneous immune responses against WT1 and Pr3 in leukemia patients are presented and the potential of WT1 and Pr3 for adoptive T-cell immunotherapy of leukemia is discussed. We also elaborate on the use of professional antigen-presenting cells loaded with mRNA encoding WT1 exploiting the advantage of broad HLA coverage for therapeutic vaccination purposes. Finally, the summarized data underscore the potential of WT1 for the manipulation of T-cell immunity in leukemia and in cancer in general, that will likely pave the way for the development of more effective and generic cancer vaccines.

Effects of interleukin-4 (IL4) on myelopoiesis: studies on highly purified CD34+ hematopoietic progenitor cells.

We studied the effects of interleukin 4 (IL4) on myelopoiesis supported by either granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) or IL3 on purified CD34+ bone marrow progenitor cells. IL4 stimulates the colony-forming unit granulocyte (CFU-G) induced by G-CSF and inhibits all colony types supported by either IL3 and GM-CSF, although inhibition of CFU-M (macrophage) was significantly stronger than that of CFU-G and CFU-GM. When the cells were first incubated in liquid culture for 4 days in IL4, followed by agar culture in G-CSF, there was a significant increase in the number of CFU-G compared to cells which had been incubated in medium alone for 4 days before plating out in agar containing G-CSF. The inhibitory effects of IL4 on GM-CSF or IL3 supported colony formation, however, disappear with sequential incubation in IL4 in liquid culture followed by culture in agar with either GM-CSF or IL3.

Karyotypic evidence for the leukemic involvement of the erythroblasts in erythroleukemia.

With the use of a monoclonal anti-glycophorin A antibody and flow cytometric cell sorting, an erythroleukemic bone marrow sample was separated in highly purified erythroblast and myeloblast fractions. Similar karyotypic anomalies were found in both cell populations as in the unseparated bone marrow. This study confirms that acute nonlymphocytic leukemia can originate at the level of a multipotential hemopoietic stem cell.

A quantitative and dynamic study of endothelial cells and megakaryocytes in human long-term bone marrow cultures.

The quantitative evolution of endothelial cells (ECs) in Dexter-type human long-term bone marrow cultures (HLTBMCs) was investigated. Using monoclonal antibodies directed against von Willebrand factor (vWF) and against membrane antigens (EN-4 and PAL-E), a low percentage--usually less than 1% of stromal cells--of ECs was detected in all confluent cultures established from 11 different bone marrow samples. Generally these cells are not associated directly with the areas of myelopoiesis ("cobblestone areas"). ECs cannot be demonstrated in the adherent layer of most young, non-confluent, and of some old, HLTBMCs. In some instances, morphological features suggestive of dynamic behavior were seen (sprouting, canal formation). In addition, a very low proportion of vWF-positive megakaryocytic cells was found in 4 of 11 cultures, always in direct contact with the stromal fibroblastic cells.

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.

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.

Stromal populations and fibrosis in human long-term bone marrow cultures.

An immunofluorescence study of the adherent layer of human long-term bone marrow cultures (HLTBMC) revealed the following surface markers on the different stromal cell populations: stromal fibroblastic cells CD10+, FIB86.3+, CD13+, CD71+; adipocytes CD10+, FIB86.3-, CD13+, CD71-/+; and macrophages CD10-/+, FIB86.3+, CD13+, CD71-/+, CD14+, CD33+, CD25+, HLA-DR+, CD4+, CD19+, CD45+. The markers of the stromal fibroblastic cells in HLTBMC were similar to those of twice-passaged fibroblasts not only from bone marrow and spleen, but also from a hemopoietic non-supportive organ such as the skin. Some of the cultured human umbilical vein endothelial cells used as controls were found to be CD25+, demonstrating for the first time the interleukin-2 receptor p55 chain on normal non-hemopoietic cells. The stromal fibroblastic cells are overrepresented compared to the small non-macrophage hemopoietic cell population in the adherent layer of HLTBMC. In addition, silver staining revealed an increased reticulin content in most of the HLTBMC. An excessive growth of stromal fibroblastic cells and an excessive deposition of their product, the reticulin fibers, are the hallmark of myelofibrosis. The finding of equivalent observations in HLTBMC suggests that the hitherto unexplained, premature quenching of hemopoiesis in HLTBMC might at least partly be due to mechanisms similar to those operating in myelofibrosis in vivo.

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.

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.

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.

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.

DFNA5: hearing impairment exon instead of hearing impairment gene?

BACKGROUND: Three mutations in the DFNA5 gene have been described in three families with autosomal dominant non-syndromic hearing impairment. Although these mutations are different at the genomic DNA level, they all lead to skipping of exon 8 at the mRNA level. We hypothesise that hearing impairment associated with DFNA5 is caused by a highly unusual mechanism, in which skipping of one specific exon leads to disease that is not caused by other mutations in this gene. We hypothesise that this represents a very specific "gain of function" mutation, with the truncated protein exerting a deleterious new function. METHODS: We performed transfection experiments in mammalian cell lines (HEK293T and COS-1) with green fluorescent protein (GFP) tagged wildtype and mutant DFNA5 and analysed cell death with flow cytometry and fluorescence microscopy. RESULTS: Post-transfection death of HEK293T cells approximately doubled when cells were transfected with mutant DFNA5-GFP compared with wildtype DFNA5-GFP. Cell death was attributed to necrotic events and not to apoptotic events. CONCLUSION: The transfection experiments in mammalian cell lines support our hypothesis that the hearing impairment associated with DFNA5 is caused by a "gain of function" mutation and that mutant DFNA5 has a deleterious new function.

Messenger RNA electroporation: an efficient tool in immunotherapy and stem cell research.

Over the last decades medicine has developed tremendously, but still many diseases are incurable. The last years, cellular (gene) therapy has become a hot topic in biomedical research for the potential treatment of cancer, AIDS and diseases involving cell loss or degeneration. Here, we will focus on two major areas within cellular therapy, cellular immunotherapy and stem cell therapy, that could benefit from the introduction of neo-expressed genes through mRNA electroporation for basic research as well as for clinical applications. For cellular immunotherapy, we will provide a state-of-the-art on loading antigen-presenting cells with antigens in the mRNA format for manipulation of T cell immunity. In the area of stem cell research, we will highlight current gene transfer methods into adult and embryonic stem cells and discuss the use of mRNA electroporation for controlling guided differentiation of stem cells into specialized cell lineages.

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.

Effects of interleukin-4 on myelopoiesis: localization of the action of IL-4 in the CD34+HLA-DR++ subset and distinction between direct and indirect effects of IL-4.

We compared the myelopoietic effects of interleukin-4 (IL-4) on CD34+HLA-DR+ and on CD34+HLA-DR++ bone marrow progenitor cells stimulated by either granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3). IL-4 stimulates G-CSF-induced colony-forming unit-granulocyte (CFU-G) and inhibits all colony types induced by GM-CSF and IL-3 in the HLA-DR++ population, but not in the HLA-DR+ population. In CD34+HLA-DR+ cells, however, a stimulation of G-CSF-supported CFU-G was also seen with sequential application of IL-4 in liquid cultures followed by G-CSF in agar cultures. In order to confirm that these are direct effects of IL-4, single-cell culture experiments were performed with CD34+HLA-DR++ cells. In these cultures IL-4 stimulates G-CSF-induced CFU-G and only inhibits colony-forming unit-macrophage (CFU-M) regardless of the CSF used to generate them.

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.

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.