Effects of Delta1 and Jagged1 on early human hematopoiesis: correlation with expression of notch signaling-related genes in CD34+ cells

It has been shown that Notch signaling mediated by ligands of both Jagged and Delta families expands the hematopoietic stem cell compartment while blocking or delaying terminal myeloid differentiation. Here we show that Delta1- and Jagged1-expressing stromal cells have distinct effects on the clonogenic and differentiation capacities of human CD34(+) CD38(+) cells. Jagged1 increases the number of bipotent colony-forming unit-granulocyte macrophage (CFU-GM) and unipotent progenitors (CFU-granulocytes and CFU-macrophages), without quantitatively affecting terminal cell differentiation, whereas Delta1 reduces the number of CFU-GM and differentiated monocytic cells. Expression analysis of genes coding for Notch receptors, Notch targets, and Notch signaling modulators in supernatant CD34(+) cells arising upon contact with Jagged1 and Delta1 shows dynamic and differential gene expression profiles over time. At early time points, modest upregulation of Notch1, Notch3, and Hes1 was observed in Jagged1-CD34(+) cells, whereas those in contact with Delta1 strikingly upregulated Notch3 and Hes1. Later, myeloid progenitors with strong clonogenic potential emerging upon contact with Jagged1 upregulated Notch1 and Deltex and downregulated Notch signaling modulators, whereas T/NK progenitors originated by Delta1 strikingly upregulated Notch3 and Deltex and, to a lesser extent, Hes1, Lunatic Fringe, and Numb. Together, the data unravel previously unrecognized expression patterns of Notch signaling-related genes in CD34(+) CD38(+) cells as they develop in Jagged1- or Delta1-stromal cell environments, which appear to reflect sequential maturational stages of CD34(+) cells into distinct cell lineages.     

HIV-1 Nef protein expression in human CD34+ progenitors impairs the differentiation of an early T/NK cell precursor

HIV-1 impairs the production of T cells, through mechanisms that are still unknown. Here, we investigated the effect of the expression of HIV-1 Nef on the T-cell potential of human hematopoietic CD34(+) precursors. Those progenitors were transduced by using lentiviral vectors expressing Nef and cultured on OP9-DL1 cells allowing the differentiation of T cell from human hematopoietic precursors. We demonstrate that Nef impairs the generation of a CD3epsilon(+)CD5(+) CD1a(+) precursor stage that has initiated a D-J rearrangement of the TCRbeta locus. Onward stages of T-cell development were also affected with a quantitative reduction of CD4(+) intraCD3epsilon(+) Immature single positive cells (ISP), Double Positive (DP) CD4(+)CD8(+) TCRalphabeta T cells and CD56(+) NK cells. But B cell production was not affected. Limiting dilution analyses demonstrated a significant reduction in the frequency of T/NK progenitors among Nef-expressing CD34(+) cells. Altogether, these data demonstrate that Nef interferes with the differentiation of a primitive lymphoid human precursor with a T/NK potential.     

Activated Notch Supports Development of Cytokine Producing NK Cells Which Are Hyporesponsive and Fail to Acquire NK Cell Effector Functions

Natural Killer (NK) cells are powerful effectors of cytotoxicity against “stressed” cells. They also produce cytokines and chemokines to activate the adaptive immune response. Understanding NK cell development and maturation may have implications for cancer therapy and for immunity against infections. We hypothesized that Notch signaling, critical for hematopoesis, would be involved in NK cell development. The role of constitutively activated Notch1 (ICN) on NK cell maturation was studied using human umbilical cord blood (UCB) progenitors cultured on a murine embryonic liver stroma cell line (EL08-1D2) and human cytokines. UCB CD34⁺/ICN⁺ sorted cells resulted in a population of CD7⁺ early lymphoid precursors and subsequent NK lineage commitment independent of stroma or IL-15. Early expression of L-selectin on ICN⁺ precursors suggested their homing competence. These precursors further committed to the NK lineage, and were capable of producing cytokines and chemokines such as interkeukin (IL)-13, granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-α), yet poorly acquired NK inhibitory receptors and cytotoxic effector function. In the presence of stroma, ICN⁺ precursors also gave rise to a population of early T lineage committed cells characterized by expression of cytoplasmic CD3 γ, ε, and δ chains, RAG1/2, and production of IL-2, suggesting bona fide Th1 commitment. Importantly, signals from EL08-1D2 stroma were required for this development process. In conclusion, sustained Notch signaling can replace stroma in differentiation of a common CD7⁺ lymphoid precursor from UCB CD34⁺ progenitors and induce NK cell commitment. However, these NK cells are immature in their cytokine production profile, are hyporesponsive, and poorly acquire NK cell receptors involved in self-tolerance and effector function.     

Notch/Delta4 interaction in human embryonic liver CD34+ CD38- cells: positive influence on BFU-E production and LTC-IC potential maintenance

We investigated whether Notch signaling pathways have a role in human developmental hematopoiesis. In situ histochemistry analysis revealed that Notch1, 2, and 4 and Notch ligand (Delta1-4, and Jagged1) proteins were not expressed in the yolk sac blood islands, the para-aortic splanchnopleure, the hematopoietic aortic clusters, and at the early stages of embryonic liver hematopoiesis. Notch1-2, and Delta4 were eventually detected in the embryonic liver, from 34 until 38 days postconception. Fluorescence-activated cell sorter analysis showed that first-trimester embryonic liver CD34(+)CD38(low) cells expressed both Notch1 and Notch2. When these cells were cultured on S17 stroma stably expressing Delta4, a 2.6-fold increase in BFU-E number was observed at day 7, as compared with cultures with control stroma, and this effect was maintained for 2 weeks. Importantly, exposure of these cells to Delta4 under these conditions maintained the original frequency and quality of long-term culture-initiating cells (LTC-ICs), while control cultures quickly resulted in the extinction of this LTC-IC potential. Furthermore, short-term exposure of embryonic liver adherent cells to erythropoietin resulted in a dose-dependent increase in Delta4 expression, almost doubling the expression observed with untreated stroma. This suggests that Delta4 has a role in the regulation of hematopoiesis after a hypoxic stress in the fetus.     

A human CD34(+) subset resides in lymph nodes and differentiates into CD56bright natural killer cells

In humans, T cells differentiate in thymus and B cells develop in bone marrow (BM), but the natural killer (NK) precursor cell(s) and site(s) of NK development are unclear. The CD56bright NK subset predominates in lymph nodes (LN) and produces abundant cytokines compared to the cytolytic CD56dim NK cell that predominates in blood. Here, we identify a novel CD34dimCD45RA(+) hematopoietic precursor cell (HPC) that is integrin alpha4beta7bright. CD34dimCD45RA(+)beta7bright HPCs constitute <1% of BM CD34(+) HPCs and approximately 6% of blood CD34(+) HPCs, but >95% of LN CD34(+) HPCs. They reside in the parafollicular T cell regions of LN with CD56bright NK cells, and when stimulated by IL-15, IL-2, or activated LN T cells, they become CD56bright NK cells. The data identify a new NK precursor and support a model of human NK development in which BM-derived CD34dimCD45RA(+)beta7bright HPCs reside in LN where endogenous cytokines drive their differentiation to CD56bright NK cells in vivo.     

Notch-induced hIL-6 production facilitates the maintenance of self-renewal of hCD34+ cord blood cells through the activation of Jak-PI3K-STAT3 pathway

Ex vivo expansion of CD34(+) stem cells in contact culture between hCD34(+)CD38(-)Lin(-) cord blood stem cells and human delta-like-expressing AFT024 feeder cells revealed increased amounts of stemness-related proteins such as HoxB4, GATA2, Bmi-1, and p21 and anti-apoptotic proteins such as Bcl-2, Bcl-xL, Mcl-1, and phospho-Bad, when compared with control or noncontact culture. Production of human IL-6 (hIL-6) was markedly elevated in the culture, but was profoundly inhibited by treatment with γ-secretase inhibitor. In addition, Notch-induced activation of STAT3 was directly involved in gene expression of hIL-6 and soluble hIL-6Rα, indicating the close linkage between Notch signaling and hIL-6 production. Furthermore, depletion of soluble hIL-6 (with hIL-6-specific antibodies) and inhibition of IL-6-mediated signals (with a Jak1 inhibitor and wortmannin) severely affected the maintenance of self-renewal of hCD34(+) cord blood cells. It was also observed that the ex vivo expanded CD34(+) cord blood cells were induced to reconstitute human immune cells in nonobese diabetic mice with severe combined immunodeficiency when compared with freshly isolated CD34(+) cord blood cells. Together, these results strongly demonstrate that Notch signaling in the "cell-to-cell contact" between hCD34(+) cord blood and delta-like-expressing AFT024 feeder cells facilitates maintenance of self-renewal of hCD34(+) cord blood cells through direct regulation of hIL-6 production.     

CD4(-)c-kit(-)CD3epsilon(-)IL-2Ralpha(+) Peyer's patch cells are a novel cell subset which secrete IL-5 in response to IL-2: implications for their role in IgA production

In this study, we examined which cell population contributes to IL-5 production by Peyer's patch (PP) cells. Thy1.2(-) fraction of PP cells, but not those of splenocytes, secreted IL-5 in response to IL-2. We found that CD3epsilon(-)IL-2Ralpha(+) cells purified from the Thy1.2(-)B220(-) fraction of PP cells secreted IL-5 when stimulated with IL-2. CD3epsilon(-)IL-2Ralpha(+) cells were subdivided into CD4(+) and CD4(-) populations or c-kit(+) and c-kit(-) populations, and only the CD4(-) and c-kit(-) CD3epsilon(-)IL-2Ralpha(+) cells secreted IL-5 in response to IL-2. CD3epsilon(-)IL-2Ralpha(+) cells did not express NK cell-markers and exhibited a lymphoid morphology. We have therefore identified CD3epsilon(-)IL-2Ralpha(+) cells as a unique lymphoid population that are not classified into conventional IL-5-producing cell populations, such as T cells, mast cells and NK cells. Depletion of CD3epsilon(-)IL-2Ralpha(+) cells from PP resulted in reduced IL-5 production. Furthermore, IgA secretion by B cells was increased when PP B cells were cocultured with CD3epsilon(-)IL-2Ralpha(+) cells. Taken together, these results suggest that the novel subset of CD4(-)c-kit(-)CD3epsilon(-)IL-2Ralpha(+) PP cells are capable of secreting a high level of IL-5 in response to IL-2, contribute markedly to IL-5 production and help IgA secretion by B cells.     

Induction of NK1.1(+) alpha beta TCR(+) T cells by bypassing TCR signals in ZAP-70 deficient mice

The mechanism of development of a unique subset of T cells, thymic NK1.1(+) alpha beta T cells, has been poorly understood. We found that the development of thymic NK1.1(+) alpha beta T cells was defective in mice deficient in ZAP-70. Instead, an accumulation of NK1.1(+) TCR beta(-) NK-like population was detected in the thymus and spleen of the ZAP-70 deficient (ZAP -/-) mouse. In the present report, we examined whether biochemical treatments that replace TCR-mediated positive selection signals could restore the generation of thymic NK1.1(+) alpha beta T cells in ZAP -/- mice using the thymus organ culture. We found that a higher concentration of phorbol ester (PMA) than that required for CD4(+) T cell generation and ionomycin induced the generation of NK1.1(+) alpha beta T cells. Phenotypic analysis of the induced NK1.1(+) alpha beta T cell population suggested that these cells expressed CD8 but not CD4 molecules, which is a different characteristic from ordinary thymic NK1.1(+) alpha beta T cells. These results suggest that differential signaling is required for the generation of mainstream T cells and thymic NK1.1(+) alpha beta T cells.     

IL-21 induces both rapid maturation of human CD34+ cell precursors towards NK cells and acquisition of surface killer Ig-like receptors

The NK cell maturation from CD34(+) Lin(-) hematopoietic cell precursors is a complex process that requires the direct contact with stromal cells and/or the synergistic effect of different cytokines. In this study we show that IL-21 is capable of inducing an accelerated NK cell maturation when added to cultures of CD34(+) Lin(-) cells isolated from human cord blood supplemented with IL-15, Flt3-L and SCF. After 25 days of culture, 50% of CD56(+) cells expressed various NK cell markers including the NKp46 and NKp30 triggering receptors, the CD94/NKG2A inhibitory receptor and CD16. At day 35, substantial fractions of NK cells expressed KIR, CD8 and CD2, i.e. surface markers expressed by mature NK cells, that are virtually undetectable in developing NK cells cultured in the absence of IL-21. Remarkably, similar to mature NK cells all these markers were included in the CD56(dim) cell fraction, while the CD56(bright) population was only composed of CD94/NKG2A(-) and CD94/NKG2A(+) cells. Thus, IL-21 allows the induction of a full NK cell maturation in vitro and offers an important tool for dissecting the molecular mechanisms involved in different steps of NK cell maturation and in the acquisition of a mature KIR repertoire.     

天花粉蛋白及其肽段诱导的免疫抑制依赖APC表面Notch配体的表达

新近发现,Notch信号途径参与调节外周成熟T细胞及其亚群的分化和功能发挥。本研究应用天花粉蛋白及其衍生肽处理骨髓来源的小鼠树突状细胞(DC),检测Notch配体家族分子的表达及DC对CD8+T细胞分泌细胞因子的影响。结果表明,天花粉蛋白或其衍生肽PB处理DC可使Notch配体Jagged1、Delta1分子表达明显增加,并改变CD8+T细胞细胞因子分泌格局,明显抑制Th1相关细胞因子IFN-γ的分泌,而Th2相关细胞因子IL-4和IL-10分泌明显增加。Notch信号的阻断剂可以部分逆转Tk及肽段的抑制作用。表明天花粉蛋白及其衍生肽可诱导一群具有抑制能力的CD8+T细胞,该作用依赖于DC表面Notch配体的表达。     

The Notch/Jagged pathway inhibits proliferation of human hematopoietic progenitors in vitro.

The cell surface receptor Notch1 is expressed on CD34+ hematopoietic precursors, whereas one of its ligands, Jagged1, is expressed on bone marrow stromal cells. To examine the role of Notch signaling in early hematopoiesis, human CD34+ cells were cultured in the presence or absence of exogenous cytokines on feeder layers that either did or did not express Jagged1. In the absence of recombinant growth factors, Jagged1 decreased myeloid colony formation by CD34+ cells, as well as 3H-thymidine incorporation and entry into S phase. In the presence of a strong cytokine signal to proliferate and mature, (interleukin 3 [IL-3] and IL-6, stem cell factor [SCF], and G-CSF), Jagged1 did not significantly alter either the fold expansion or the types of colonies formed by CD34+ cells. However, in the presence of SCF alone, Jagged1 increased erythroid colony formation twofold. These results demonstrate that Notch can modulate a growth factor signal, and that in the absence of growth factor stimulation, the Jagged1-Notch pathway preserves CD34+ cells in an immature state.     

Notch ligands potentiate IL‐7‐driven proliferation and survival of human thymocyte precursors

Notch and IL‐7 are both well‐characterized factors involved in T‐cell development. In contrast to the mouse model, their precise requirements in the differentiation and/or proliferation of various stages of human thymic development have not been fully explored. Here, we demonstrate that IL‐7 alone is sufficient to induce the differentiation of ex vivo purified CD34⁺ triple negative (TN) surface (s) CD3^? CD4^?CD8^? (CD3^?CD4^?CD8^?), CD4 immature single positive (ISP) (sCD3^?CD4⁺CD8^?) and double positive (DP) (sCD3^?CD4⁺CD8⁺) human thymic precursors to mature DP expressing sCD3 (sCD3⁺CD4⁺CD8⁺). We show that activation of Notch signaling by its ligands Delta‐1 or Delta‐4 potentiates IL‐7‐driven proliferation and survival of CD34⁺ TN and to a lesser extent of CD4⁺ ISP precursors. This effect of Notch is related to a sustained induction of IL‐7 receptor α chain expression on thymocytes through a decreased methylation of its gene promoter. Thus, we show here that proliferation and differentiation of T‐cell precursors are differentially modulated by IL‐7 depending on the presence or absence of external signals. These results may have important implications for the clinical use of this cytokine as a strategy aimed at improving immune restoration.     

Impaired NK cell differentiation of blood-derived CD34+ progenitors from patients with myeloid metaplasia with myelofibrosis

Cultured blood CD34(+) progenitors from patients with myeloid metaplasia with myelofibrosis (MMM) failed to differentiate into natural killer (NK) cells with recombinant interleukin (IL)-15. No NK cells either could be induced in coculture with IL-15-expressing fibroblasts from MMM patients' spleens. The impaired NK differentiation could be circumvented by using normal blood CD34(+) cells in the coculture. In this case, cell-to-cell contact and IL-15 interaction were crucial for NK cell differentiation. Pretreatment of normal CD34(+) progenitors with anti-IL-15 monoclonal antibody markedly reduced NK cell production while MMM fibroblast pretreatment did not. Both normal and MMM progenitors constitutively expressed IL-15. Analysis of endogenous IL-15 signaling pathway revealed a constitutive gammac/Jak3 association and STAT3 activation in the two types of progenitors. Anti-IL-15 monoclonal antibody treatment caused a downregulation of IL-15 signaling in normal but not MMM blood cells. The impaired NK differentiation in MMM may thus arise from a deregulated control of an endogenous IL-15 involved in hematopoietic progenitors' lymphoid differentiation.     

Notch信号介导共培养脐带间充质干细胞和造血干细胞的相互作用

目的:探讨脐带间充质干细胞(UC-MSC)体外与造血干细胞共培养后Notch信号分子的改变。方法:通过胶原酶消化方法分离UC-MSC,通过流式细胞仪检测以及成脂、成骨和成软骨诱导鉴定UC-MSC具备间充质干细胞的特性。进而,将UC-MSC与脐血CD34+造血干细胞(HSC)体外培养,实时PCR方法检测MSC及CD34+细胞表面Notch配体及受体表达以及表达是否存在变化;在共培养体系中加入Notch信号阻滞剂DAPT(γ-secretase抑制剂),比较Hes-1基因活化状态的改变。结果:体外实验显示:UC-MSC在形态学、细胞表面表型和诱导分化能力上均具备间充质干细胞的特性。UC-MSC及CD34+细胞表面存在Notch信号配体及受体的表达,共培养后Jagged 1、Notch1基因表达明显增加;共培养后CD34+细胞中的Hes-1基因表达明显增加而加入DAPT后Hes-1基因表达未检出明显改变。结论:UC-MSC支持造血中,Notch信号可能发挥重要作用。     

Delta‐like 4‐mediated Notch signaling is required for early T‐cell development in a three‐dimensional thymic structure

Delta‐like 4 (Dll4)‐mediated Notch signaling is critical for specifying T‐cell fate, but how Dll4‐mediated Notch signaling actually contributes to T‐cell development in the thymus remains unclear. To explore this mechanism in the thymic three‐dimensional structure, we performed fetal thymus organ culture using Dll4‐deficient mice. DN1a/b+DN2mt cells, which had not yet committed to either the αβ T or γδ T/NK cell lineage, did not differentiate into the αβ T‐cell lineage in Dll4‐deficient thymus despite the lack of cell fate conversion into other lineages. However, DN3 cells efficiently differentiated into a later developmental stage of αβ T cells, the double‐positive (DP) stage, although the proliferation was significantly impaired during the differentiation process. These findings suggest that the requirement for Notch signaling differs between the earliest and pre‐TCR‐bearing precursors and that continued Notch signaling is required for proper differentiation with active proliferation of αβ T lineage cells. Furthermore, we showed that Notch signaling increased the c‐Myc expression in DN3 cells in the thymus and that its overexpression rescued the proliferation and differentiation of DN3 cells in the Dll4‐null thymus. Therefore, c‐Myc plays a central role in the transition from stage DN3 to DP as a downstream target of Notch signaling.