ALBERT

Description: Although well characterized in the mouse, the role of Notch signaling in the human T-cell receptor αβ (TCR-αβ) versus TCR-γδ lineage decision is still unclear. Although it is clear in the mouse that TCR-γδ development is less Notch dependent compared with TCR-αβ differentiation, retroviral overexpression studies in human have suggested an opposing role for Notch during human T-cell development. Using the OP9-coculture system, we demonstrate that changes in Notch activation are differentially required during human T-cell development. High Notch activation promotes the generation of T-lineage precursors and γδ T cells but inhibits differentiation toward the αβ lineage. Reducing the amount of Notch activation rescues αβ-lineage differentiation, also at the single-cell level. Gene expression analysis suggests that this is mediated by differential sensitivities of Notch target genes in response to changes in Notch activation. High Notch activity increases DTX1, NRARP, and RUNX3 expression, genes that are down-regulated during αβ-lineage differentiation. Furthermore, increased interleukin-7 levels cannot compensate for the Notch dependent TCR-γδ development. Our results reveal stage-dependent molecular changes in Notch signaling that are critical for normal human T-cell development and reveal fundamental molecular differences between mouse and human.

Description: Homeobox genes are well known for their crucial role during embryogenesis but have also been found to be critically involved in normal and leukemic hematopoiesis. Because most previous studies focused on the role of aberrant HOX gene expression in leukemogenesis and because HOX-A10 is expressed in human CD34+ precursor cells, this study investigated whetherHOX-A10 also plays a pivotal role in normal hematopoietic-lineage determination. The effect of enforced expression of this transcription factor on hematopoietic differentiation of highly purified human cord-blood progenitors was examined by using in vitro assays. In fetal thymic organ cultures, a dramatic reduction in cells expressing high levels of HOX-A10 was observed, along with absence of thymocytes positive for CD3+ T-cell receptor αβ. Furthermore, in MS-5 stromal cell cultures, there was a 7-fold reduction in the number of natural killer cells and a 9-fold reduction in the number of B cells, thus showing a profound defect in differentiation toward the lymphoid lineage inHOX-A10–transduced progenitors. In contrast, the number of CD14+ monocytic cells in the stromal cell culture was 6-fold higher, suggesting an enhanced differentiation toward the myeloid differentiation pathway of HOX-A10–transduced progenitors. However, there was a slight reduction in the number of CD15+ granulocytic cells, which were blocked in their final maturation. These data show that HOX-A10 can act as an important key regulator of lineage determination in human hematopoietic progenitor cells.

Description: Human umbilical cord blood (UCB) hematopoietic stem cells (HSC) receive increased attention as a possible target for gene-transfer in gene therapy trials. Diseases affecting the lymphoid lineage, as adenosine deaminase (ADA) deficiency and acquired immunodeficiency syndrome (AIDS) could be cured by gene therapy. However, the T-cell progenitor potential of these HSC after gene-transfer is largely unknown and was up to now not testable in vitro. We show here that highly purified CD34++ Lineage marker-negative (CD34++Lin−) UCB cells generate T, natural killer (NK), and dendritic cells in a severe combined immunodeficient mouse fetal thymus organ culture (FTOC). CD34++Lin− and CD34++CD38−Lin− UCB cells express the retroviral encoded marker gene Green Fluorescent Protein (GFP) after in vitro transduction with MFG-GFP retroviral supernatant. Transduced cells were still capable of generating T, NK, and dendritic cells in the FTOC, all expressing high levels of GFP under control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat promotor. We thus present an in vitro assay for thymic T-cell development out of transduced UCB HSC, using GFP as a marker gene.

Description: Notch receptors are involved in lineage decisions in multiple developmental scenarios, including hematopoiesis. Here, we treated hybrid human-mouse fetal thymus organ culture with the γ-secretase inhibitor 7 (N-[N-(3,5-difluorophenyl)-l-alanyl]-S-phenyl-glycine t-butyl ester) (DAPT) to establish the role of Notch signaling in human hematopoietic lineage decisions. The effect of inhibition of Notch signaling was studied starting from cord blood CD34+ or thymic CD34+CD1-, CD34+CD1+, or CD4ISP progenitors. Treatment of cord blood CD34+ cells with low DAPT concentrations results in aberrant CD4ISP and CD4/CD8 double-positive (DP) thymocytes, which are negative for intracellular T-cell receptor β (TCRβ). On culture with intermediate and high DAPT concentrations, thymic CD34+CD1- cells still generate aberrant intracellular TCRβ- DP cells that have undergone DJ but not VDJ recombination. Inhibition of Notch signaling shifts differentiation into non-T cells in a thymic microenvironment, depending on the starting progenitor cells: thymic CD34+CD1+ cells do not generate non-T cells, thymic CD34+CD1- cells generate NK cells and monocytic/dendritic cells, and cord blood CD34+Lin- cells generate B, NK, and monocytic/dendritic cells in the presence of DAPT. Our data indicate that Notch signaling is crucial to direct human progenitor cells into the T-cell lineage, whereas it has a negative impact on B, NK, and monocytic/dendritic cell generation in a dose-dependent fashion.

Description: In vitro generation of mature T cells from human hematopoietic stem and progenitor cells (HSPC) could fulfill two existing needs. First, it could enhance and quicken T cell immune reconstitution after stem cell transplantation, which is very slow and generates a skewed TCR repertoire. Second, by generation of tumour antigen specific T cells it could provide an efficient therapy for numerous malignancies and could enhance GVT effect in the context of allogeneic SCT, without aggravating GVHD. T cells can be generated from human HSPC by culturing them on the murine stromal cell line OP9-transduced with the Notch ligand Delta-like-1 (OP9-DL1). Notch receptor activation is essential for T cell development. However, it is unclear whether Notch activation is sufficient for end maturation into functionally and phenotypically mature TCR positive cells. It was shown that human CD34+ cells cultured on OP9-DL1 differentiate to T cells which can proliferate and produce interferon-g upon polyclonal stimulation. The nature of the mature cells generated in these cultures, however, has not been well studied. CD34+ HSPC from postnatal thymus (PNT) or cord blood were cocultured with OP9-DL1, in the presence of the cytokines Flt-3L (5 ng/ml), SCF (2.5 ng/ml) and IL-7 (5 ng/ml). Every 3–5 days cells were harvested and transferred to fresh OP9-DL1 cells. At repetitive timepoints, an aliquot of the cells was analysed phenotypically. In some experiments, IL-15 was added to the culture. For some experiments, cells harvested from OP9-DL1 at the timepoint mature T cells were observed (usually about d 40 of culture), were transferred to feeder cells, consisting of JY cell line (5.104 cells/ml irradiated with 50 Gy and PBMC (5.105/ml irradiated with 40 Gy), in the presence of PHA (1 mg/ml). After 7 days, IL-2 (50 IU/ml) was added to the culture. Every 14 days, cells were restimulated with new feeders (irradiated JY and PBMC) and new addition of PHA. After 3 weeks of stimulation cells were stimulated overnight with 15 ng/ml PMA and 1500 ng/ml ionomycin, and 18 hours later cells were checked for intracellular presence of cytokines. We investigated whether the T cell population generated in these cultures contains mature cells with the characteristics of TCRγδ cells and of positively selected CD8 or CD4 single positive (SP) TCRαβ cells as observed in the human thymus. We found that under the described conditions, HSPC mature into CD1-CD27+ phenotypically mature T cells, with the TCRγδ fraction maturing faster and more efficiently compared to the TCRαβ fraction. Consistent with a mature phenotype, TCRγδ cells were mostly CD8αα or double negative (DN). No mature CD4 SP TCRαβ cells were observed and the mature CD8 SP cells co-expressed variable ratios of CD8αβ and CD8αα dimers, suggesting that these cells are not conventional positively selected TCRαβ cells. In support of this hypothesis, both mature CD1- TCRαβ and TCRγδ cells expressed the IL2Rβ receptor consitutively and both populations proliferated on IL-15 without prior antigen stimulation, CD8αα (TCRαβ and TCRγδ) cells being the most IL-15 responsive. Mature activated T cells secreted IFN-γ and TNFα, little or no IL-2 and IL-4, with no difference observed between TCRαβ and TCRγδ cells. These data suggest that CD8 TCRαβ cells generated in these cultures are unconventional CD8 cells possibly maturated through agonist selection. However, when cells harvested after 40 days of culture on OP9-DL1 were stimulated with PHA and IL-2 for 3 weeks, conventional appearing CD8αβ cells emerged, with a cytokine production profile similar to that of thymic CD8αβ TCRαβ T cells, with the majority of cells secreting IFN-γ and IL-2. We can conclude from these data that OP9-DL1 supports the development of both unconventional and conventional CD8+ TCRαβ cells, of which the generation and selection process are currently being investigated. Also the in vitro anti-tumor capacities of both populations need to be addressed.

Description: Endothelial outgrowth cells (EOC) can be generated from mononuclear blood cells. Based on proliferative and functional characteristics, EOC were claimed to derive from an immature endothelial progenitor cell or angioblast. Several investigators have claimed that these cells constitute a subpopulation of CD34+ hematopoietic stem cells(HSC). However, the EOC-precursor is not well defined and its nature remains elusive. Methods and results: Umbilical cord blood CD34+ cells were sorted into a small (〈 1 %) CD34+CD45− non-hematopoietic cell fraction (purity 〉 99.5%) and CD34+CD45+ HSC (purity 〉 99.2 %) (n=5). The cell fractions were cultured separately in EBM2/EGM2 medium (Cambrex, Verviers, Belgium) onto gelatine coated 24 wells. EOC were exclusively derived from the CD34+CD45− cell fraction and not from the CD45+ HSC. We further analysed the CD34+CD45− cell fraction for expression of endothelial progenitor genes. Analysis showed the presence of VEGFR2, VE-Cadherine and CD146 on the CD34+CD45− precursor population whereas CD45+ HSC were consistantly negative for these markers. CD133, which was claimed to be a marker for endothelial progenitors was negative on the CD34+CD45− cells. No VEGFR2+ CD133+ cells could be detected either by flowcytometry or at the mRNA level. In adult bone marrow, EOC only derived from CD45− CD31+ cells, and not from the CD45+ HSC or CD45− CD31− mesenchymal cells. CD34+CD45+ HSC or CD14+ CD45+ monocytes generated under the same conditions large flat adherent cells positive for CD31, LDL uptake and the lectin UEA-1. On RT-PCR and real time RT-PCR analysis, cells were positive for VEGFRII, CD146 and VE cadherin. However, membrane staining was consistently negative for VE-cadherin on flowcytometric analysis and positive for monocytic markers such as CD14 and CD45. In functional assays, the majority of the cells were shown to be phagocytic and were unable to form vascular tubes in the matrigel angiogenesis assay. These data demonstrate that monocytes may acquire a phenotype in vitro which is difficult to discriminate from endothelial cells. Conclusion : Endothelial cell generated in vitro from cord blood or bone marrow derive from a CD45− nonhematopoietic precursor.

Description: 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)- 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- effect resulted in an up-regulation of CD127 (ie, the IL-7 receptor -chain) in a small subset of the CD34+ cells. No evidence could be generated to support the possibility that TNF- 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.

Description: Notch signaling critically mediates various hematopoietic lineage decisions and is induced in mammals by Notch ligands that are classified into 2 families, Delta-like (Delta-like-1, -3 and -4) and Jagged (Jagged1 and Jagged2), based on structural homology with both Drosophila ligands Delta and Serrate, respectively. Because the functional differences between mammalian Notch ligands were still unclear, we have investigated their influence on early human hematopoiesis and show that Jagged2 affects hematopoietic lineage decisions very similarly as Delta-like-1 and -4, but very different from Jagged1. OP9 coculture experiments revealed that Jagged2, like Delta-like ligands, induces T-lineage differentiation and inhibits B-cell and myeloid development. However, dose-dependent Notch activation studies, gene expression analysis, and promoter activation assays indicated that Jagged2 is a weaker Notch1-activator compared with the Delta-like ligands, revealing a Notch1 specific signal strength hierarchy for mammalian Notch ligands. Strikingly, Lunatic-Fringe– mediated glycosylation of Notch1 potentiated Notch signaling through Delta-like ligands and also Jagged2, in contrast to Jagged1. Thus, our results reveal a unique role for Jagged1 in preventing the induction of T-lineage differentiation in hematopoietic stem cells and show an unexpected functional similarity between Jagged2 and the Delta-like ligands.

Description: Thymic repopulation by transplanted hematopoietic progenitor cells (HPC) is likely to be important for long-term immune reconstitution and for successful gene therapy of diseases affecting the T-cell lineage. However, the T-cell progenitor potential of HPC, cultured in vitro for cell number expansion and gene transfer remains largely unknown. Here, we cultured highly purified human umbilical cord blood (CB) CD34+CD38− or CD34+CD38+ cells for up to 5 weeks in stroma-free cultures supplemented with various combinations of the cytokines thrombopoietin (TPO), stem cell factor (SCF), flt3/flk-2 ligand (FL), interleukin-3 (IL-3), and IL-6 and investigated thymus-repopulating ability of expanded cells in vitro and in vivo. After up to 5 weeks of culture in IL-3 + SCF + IL-6 or TPO + FL + SCF supplemented medium, the progeny of CD34+CD38− CB cells generated T cells and natural killer cells in the thymus. Limiting dilution experiments demonstrated increase in the number of T-cell progenitors during culture. After 3 weeks of culture, gene marked CD34+CD38− CB cells injected in the human thymus fragment transplanted in severe combined immunodeficient (SCID) mice (SCID-hu) generated thymocytes expressing the retroviral encoded marker gene GFP in vivo. Thus, our results show that the progeny of CD34+CD38− CB cells cultured for extensive periods, harbor thymus-repopulating cells that retain T-cell progenitor potential after expansion and gene transfer.