D. R. Van Bockstaele

Generation of dendritic cells from bone marrow progenitors using GM-CSF, TNF-alpha, and additional cytokines: antagonistic effects of IL-4 and IFN-gamma and selective involvement of TNF-alpha receptor-1.

We report the generation of dendritic cells (DC) starting from CD34+ bone marrow (BM) progenitor cells, using a two‐stage culture system in which, besides granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and tumour necrosis factor‐α (TNF‐α), stem‐cell factor (SCF) was added during the first 5 days, while interleukin‐4 (IL‐4) and/or interferon‐γ (IFN‐γ) were added during the secondary culture period of 9 days. Addition of IL‐4 favoured the outgrowth of CD1a+, HLA‐DR+, CD4+, CD40+, CD80+ but CD14− cells with dendritic morphology and strong antigen‐presenting capacity. Addition of IFN‐γ selectively induced HLA‐DR and CD86 but did not up‐regulate CD1a expression or antigen‐presenting capacity of the differentiated cells. An antagonism between IL‐4 and IFN‐γ could further be confirmed in that, as compared with IL‐4 alone, the simultaneous addition of IL‐4 and IFN‐γ to GM‐CSF plus TNF‐α during maturation reduced both the phenotypical (CD1a, CD4, CD40) and functional characteristics of DC. Using receptor‐specific TNF‐α mutants, we investigated the relative involvement of TNF‐α receptors R1 and R2 in the generation of DC. The induction of CD1a and HLA‐DR, as well as the increase in allostimulatory capacity were dependent on TNF‐R1 triggering, whereas triggering through TNF‐R2 had no measurable effect. We conclude first, that the expansion of DC from BM progenitors could most effectively be enhanced in a two‐stage culture assay using SCF, GM‐CSF, TNF‐α and IL‐4; second, that the effect of TNF‐α in DC generation involves signalling via the TNF‐R1 receptor; and third, that IFN‐γ counteracts some of the effects of IL‐4 in DC generation.

Immunosuppression induced by immature dendritic cells is mediated by TGF‐β/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)‐β 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‐β 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‐β+/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‐β+/IL‐10+ T cells are expanded by DC independent of their maturation status, but their suppressive function is dependent on immaturity of DC.

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.

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.

A myelodysplastic syndrome preceding acute lymphoblastic leukaemia

The bone marrow of a patient with pancytopenia showed dyserythro‐poiesis, dysmegakaryocytopoiesis and 13% blasts. The patient was hypertransfused and the pancytopenia resolved completely for 1 month, while the blastic infiltration in the bone marrow remained. Three months later a frank acute lymphoblastic leukaemia developed.

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.

Direct effects of 13-cis and all-trans retinoic acid on normal bone marrow (BM) progenitors: Comparative study on BM mononuclear cells and on isolated CD 34+ BM cells

SummaryThe effects of both 13-cis-and all-trans retinoic acid (RA) on colony formation of normal bone marrow (BM) progenitors were investigated in semi-solid (methylcellulose) assays, using either isolated CD 34+ cells or BM mononuclear cells. Single cell liquid cultures were performed to further discriminate between direct and indirect effects. RA action results in significant decrease of colony forming units (CFUs). This effect is more pronounced starting from CD 34+ progenitors than starting from total BM. This overall decrease in CFUs is due to selective inhibition of CFU-M (macrophage) and erythroid colonies (BFU-E). At the single cell level the CFU-M inhibition is confirmed with — in addition — a significant inhibition of CFU-GM (granulocyte-macrophage) and a marked stimulation of CFU-G(granulocyte)s. Both retinoids exert the above-mentioned effects. All-trans RA, however, is effective at a tenfold lower concentration (10−7M) than 13-cis RA (10−6M). Results on CD 34+ BM fractions (substantially reduced in accessory cells) demonstrate that the described effects can probably be attributed to the direct action of RA on these progenitors; single progenitor (CD 34+) cell liquid cultures further prove this point.

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.

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.

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.