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.     

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.     

The interaction of the Wnt and Notch pathways modulates natural killer versus T cell differentiation

The Wnt and Notch signaling pathways have been independently shown to play a critical role in regulating hematopoietic cell fate decisions. We previously reported that induction of Notch signaling in human CD34(+)CD38(-) cord blood cells by culture with the Notch ligand Delta 1 resulted in more cells with T or natural killer (NK) lymphoid precursor phenotype. Here, we show that addition of Wnt3a to Delta 1 further increased the percentage of CD34(-)CD7(+) and CD34(-)CD7(+)cyCD3(+) cells with increased expression of CD3 epsilon and preT alpha. In contrast, culture with Wnt3a alone did not increase generation of CD34(-)CD7(+) precursors or expression of CD3 epsilon or preT alpha gene. Furthermore, Wnt3a increased the amount of activated Notch1, suggesting that Wnt modulates Notch signaling by affecting Notch protein levels. In contrast, addition of a Wnt signaling inhibitor to Delta 1 increased the percentage of CD56(+) NK cells. Overall, these results demonstrate that regulation of Notch signaling by the Wnt pathway plays a critical role in differentiation of precursors along the early T or NK differentiation pathways. Disclosure of potential conflicts of interest is found at the end of this article.     

HES1 inhibits cycling of hematopoietic progenitor cells via DNA binding

Notch signaling is implicated in stem cell self-renewal, differentiation, and other developmental processes, and the Drosophila hairy and enhancer of split (HES) 1 basic helix-loop-helix protein is a major downstream effector in the Notch pathway. We found that HES1 was expressed at high levels in the hematopoietic stem cell (HSC)-enriched CD34+/[CD38/Lin](- /low) subpopulation but at low levels in more mature progenitor cell populations. When CD34+ cells were cultured for 1 week, the level of HES1 remained high in the CD34+ subset that had remained quiescent during ex vivo culture but was reduced in CD34+ cells that had divided. To investigate the effects of HES1 in human and mouse hematopoietic stem-progenitor cells (HSPCs), we constructed conditional lentiviral vectors (lentivectors) to introduce transgenes encoding either wild-type HES1 or a mutant lacking the DNA-binding domain (BHES1). We found that lentivector-mediated HES1 expression in CD34+ cells inhibited cell cycling in vitro and cell expansion in vivo, associated with upregulation of the cell cycle inhibitor p21(cip1/Waf1) (p21). The HES1 DNA-binding domain was required for these actions. HES1 did not induce programmed cell death or alter differentiation in HSPCs, and while short-term repopulating activity was reduced in HES1-transduced mouse and human cells, long-term reconstituting HSC function was preserved. Our data characterize the complex, cell context-dependent actions of HES1 as a major downstream Notch signaling regulator of HSPC function.     

T-Cell Immune Imbalance in Rheumatoid Arthritis Is Associated with Alterations in NK Cells and NK-Like T Cells Expressing CD38

BackgroundCD38+ NK (CD3− CD16+ CD38+ CD56+) cells were increased in rheumatoid arthritis (RA), which suppressed Treg cell differentiation. This study explored how CD38+ NK cells regulated CD4+ T-cell differentiation into Treg cells in RA.MethodsProportions of CD38+ NK cells and their counterpart CD38+ NK-like T (CD3+ CD16+ CD38+ CD56+) cells were measured in RA and rats with collagen-induced arthritis (CIA). CD38+ NK cells and CD38+ NK-like T cells were cocultured with CD4+ T cells, respectively.ResultsA significantly increased proportion of CD38+ NK cells and a decreased proportion of CD38+ NK-like T cells were detected in RA and CIA blood and synovial fluids. When CD4+ T cells were cocultured with CD38+ NK cells, mammalian target of rapamycin (mTOR) signaling was activated, and Th1/Th2 and Th17/Treg ratios were increased. When CD38+ NK cells were pretreated with anti-CD38 antibody, Treg cell proportion was increased, and Th1/Th2 and Th17/Treg ratios were decreased. CD38+ NK-like T cells showed the opposite results. CD38+ NK cells and CD38+ NK-like-T cells activated differential gene expressions and pathways in CD4+ T cells and initiated Th1 and Th2 cell differentiation by differential gene nodes.ConclusionsThis study suggest that the high CD38+ NK cell proportion and low CD38+ NK-like T cell proportion in RA suppress Treg cell differentiation by stimulating mTOR signaling in CD4+ T cells, which consequentially disturbs the immune tolerance.     

Notch1 regulates maturation of CD4+ and CD8+ thymocytes by modulating TCR signal strength

Notch signaling regulates cell fate decisions in multiple lineages. We demonstrate in this report that retroviral expression of activated Notch1 in mouse thymocytes abrogates differentiation of immature CD4+CD8+ thymocytes into both CD4 and CD8 mature single-positive T cells. The ability of Notch1 to inhibit T cell development was observed in vitro and in vivo with both normal and TCR transgenic thymocytes. Notch1-mediated developmental arrest was dose dependent and was associated with impaired thymocyte responses to TCR stimulation. Notch1 also inhibited TCR-mediated signaling in Jurkat T cells. These data indicate that constitutively active Notch1 abrogates CD4+ and CD8+ maturation by interfering with TCR signal strength and provide an explanation for the physiological regulation of Notch expression during thymocyte development.     

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。     

Activation of the Notch signaling pathway disturbs the CD4+/CD8+, Th17/Treg balance in rats with experimental autoimmune uveitis

Objective and design

The present study aimed to investigate the relationship between the disturbed balance of CD4⁺/CD8⁺, Th17/Treg and the activation of the Notch signaling pathway in experimental autoimmune uveitis (EAU).

Methods

An EAU rat model was induced in Lewis rats, and pathology analysis was performed by hematoxylin and eosin (H&E) staining. CD4⁺, CD8⁺, Th17, and Treg levels in spleen, lymph nodes and eye tissues were determined by flow cytometry. Meanwhile, the expression of Notch1, DLL4, IL-10, and IL-17 was determined by quantitative polymerase chain reaction (Q-PCR) and enzyme-linked immunosorbent assay (ELISA). In addition, the inhibitory effect of N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT) on Th17 differentiation by Notch signaling in vitro was further investigated using T lymphocytes from EAU rats on day 12 post-immunization by flow cytometry.

Results

The pathological results showed that inflammatory cell infiltration occurred in ocular tissues in EAU rats. The CD4⁺/CD8⁺ and Th17/Treg ratios in EAU rats were apparently higher than those in normal control individuals. Q-PCR and ELISA analyses indicated the expression of Notch1, DLL4, IL-10, and IL-17 in EAU rats gradually increased on day 6 after immunization, peaked on day 12, and then gradually decreased. The dynamic trends in Notch1 and DLL4 expression in EAU rats were identical to those of CD4⁺/CD8⁺ and Th17/Treg levels. DAPT can significantly inhibit the activation of Notch signaling, decrease Th17 cell differentiation, and attenuate the level of the Th17 cell lineage, contributing to the balance of the Th17/Treg ratio.

Conclusion

The activation of the Notch signaling pathway can regulate Th17 and Treg cell differentiation, disrupt the CD4⁺/CD8⁺ and Th17/Treg balance, and aggravate the severity of EAU; inactivation of the Notch signaling pathway contributes to the CD4⁺/CD8⁺ and Th17/Treg balance in EAU rats. Our findings highlighted that the dynamic change in the CD4⁺/CD8⁺ and Th17/Treg ratio was consistent with the expression trend of Notch signaling in EAU rats, suggesting that Notch signaling may be a potentially important therapeutic target in clinical practice.

     

Production of human B cells from CD34+CD38- T- B- progenitors in organ culture by sequential cytokine stimulation

We investigated sequential cytokine addition on human hematopoietic stem cell (HSC) differentiation in murine fetal liver (FL), fetal spleen (FS) and bone marrow (BM) organ cultures (OC). Tissues were colonized with unpurified or FACS sorted CD34+CD38-CD10-CD19-CD3-CD8-CD4-(T- B-) cells from human cord blood (HUCB). CD19+ cell production and kinetics differed in each tissue. Fetal liver organ cultures (FLOC) inoculated with CD34+CD38-T-B- cells produced fewer CD19+ cells than fetal liver organ culture (FLOC) cultured with unpurified HUCB. CD19+ cell production was restored in the CD34+CD38-T-B- organ cultures by treating with SCF, LIF and IL-6 followed by IL-7 and removing all cytokines for the last 3 days of culture (a six-fold increase). FLOC also produced CD34+CD38-T-B- cells and monocyte-lineage CD33+CD14- cells, both of which increased after cytokine treatment. Re-colonization of secondary FLOC with CD34+CD38-T-B- cells generated in primary FLOC produced additional B-cells, monocytes and CD34+CD38- cells suggesting that the primary cells retained HSC activity. Expansion and differentiation of HSCs depended on the microenvironment of the recipient tissue as well as addition of cytokines in the appropriate order.     

Notch signalling regulates cytokine production by CD8+ and CD4+ T cells

The Notch signalling pathway regulates several aspects of cellular differentiation such as T lineage commitment and effector functions on peripheral T cells; however, there is limited information regarding Notch receptor expression on different T cell subsets and the putative role of the different receptors on T cell effector function. Here, we studied the protein expression of Notch receptors on murine T cells in vitro and in vivo and analysed the role of the Notch pathway in cytokine production by CD4+ and CD8+ T cells. We found that resting CD4+ and CD8+ T cells do not express Notch receptors, but they upregulate Notch 1 and Notch 2 shortly after in vitro and in vivo activation. Using a γ-secretase inhibitor, which blocks Notch signalling through all Notch receptors, we demonstrated that the Notch pathway regulates IL-10 production by CD4+ T cells and IFN-γ and IL-17 production by CD8+ T cells. These results suggest that Notch 1 and 2 are expressed by CD4+ and CD8+ T cells and represent the putative Notch receptors that regulate effector functions and cytokine production by these cells.     

Catalytic activities of G1 cyclin-dependent kinases and phosphorylation of retinoblastoma protein in mobilized peripheral blood CD34+ hematopoietic progenitor cells

Depending on the source of cells, the cell cycle status of hematopoietic stem and progenitor cells capable of repopulating the marrow of transplant recipients is controversial. In this study, using biochemical methods, the cell cycle status of mobilized CD34+ cells was analyzed. It was demonstrated in CD34+ cell extracts that there was high catalytic activity of G(1) cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) but low activity of CDK2. This was in contrast to the resting reference cells that showed only minimal or no activity of these CDKs. Since at the G0-->G1-->S transition CDK4/6 and CDK2 sequentially phosphorylate the retinoblastoma protein (pRB), its phosphorylation status was analyzed. Previously, we showed that p110RB was unphosphorylated at serine (Ser)-608 in CD34+ cells, consistent with the ability to suppress cell growth. Here, it was established that this form of pRB was phosphorylated at Ser-780, Ser-795, and Ser-807/811 in CD34+ but not in resting reference cells. This result was therefore consistent with the presence of high CDK4/6 activities in CD34+ cells. Conversely, CDK2 activity was low and the pRB residues Ser-612 and threonine (Thr)-821, which are exclusively phosphorylated by CDK2 in conjunction with either cyclin E or A, were unphosphorylated in >90% of CD34+ cells. We therefore show for the first time the exact position of mobilized CD34+ cells within the cell cycle; that is, they do not reside in G0 but in early G1 phase and did not cross the restriction point into late G1 phase.     

Linked suppression in peripheral T cell tolerance to the house dust mite derived allergen Der p 1

BACKGROUND: Peripheral tolerance is required to maintain balance within the immune system. A feature of peripheral tolerance is linked suppression, in which tolerance induced to a single T cell epitope inhibits the response to all epitopes in the same protein. It is suggested that this phenomenon is mediated by regulatory T cells through either the activity of immunopressive cytokines or direct cell contact. In previous experiments we failed to detect inhibitory cytokines when T cells from mice rendered tolerant by intranasal delivery of the immunodominant peptide of Der p 1 (p 1, 110-131) were restimulated with peptide in vitro. Therefore, the aim of this study was to determine if cognate interactions between T cells mediated by Notch/Delta signaling induce and maintain peripheral T cell tolerance. METHODS: Using in situ hybridization and viral mediated gene transfer, the expression and function of Delta1 were investigated in a murine model of T cell tolerance to Der p 1 in vivo. RESULTS: Delta1 expression is increased on peripheral T cells during the induction of tolerance with high-dose peptide delivered intranasally and when tolerant animals are rechallenged under immunogenic conditions. Peptide p 1, 110-131-specific CD4+ T cells transfected with Delta1 inhibited the response of antigen-primed T cells and induced linked suppression. CONCLUSIONS: High-dose peptide delivered intranasally induces transient expression of Delta 1 on inhibitory CD4+ T cells. Ligation of the Notch1 receptor on neighbouring T cells by Delta1+ regulatory T cells inhibits clonal expansion of the former and mediates linked suppression.     

Cycling G1 CD34+/CD38+ cells potentiate the motility and engraftment of quiescent G0 CD34+/CD38-/low severe combined immunodeficiency repopulating cells

The mechanism of human stem cell expansion ex vivo is not fully understood. Furthermore, little is known about the mechanisms of human stem cell homing/repopulation and the role that differentiating progenitor cells may play in these processes. We report that 2- to 3-day in vitro cytokine stimulation of human cord blood CD34(+)-enriched cells induces the production of short-term repopulating, cycling G1 CD34(+)/CD38(+) cells with increased matrix metalloproteinase (MMP)-9 secretion as well as increased migration capacity to the chemokine stromal cell-derived factor-1 (SDF-1) and homing to the bone marrow of irradiated nonobese diabetic severe/combined immunodeficiency (NOD/SCID) mice. These cycling G1 cells enhance SDF-1-mediated in vitro migration and in vivo homing of quiescent G0 CD34(+) cells, which is partially abrogated after inhibition of MMP-2/-9 activity. Moreover, the engraftment potential of quiescent G0 SCID repopulating cells (SRCs) is also increased by the cycling G1 CD34(+)/CD38(+) cells. This effect is significantly abrogated after incubation of cycling G1 cells with a neutralizing anti-CXCR4 antibody. Our data suggest synergistic interactions between accessory cycling G1 CD34(+)/CD38(+) committed progenitor cells and quiescent, primitive G0 CD34(+)/CD38(-/low) SRC/stem cells, the former increasing the motility and engraftment potential of the latter, partly via secretion of MMP-9.     

Role of the CD34+ 38- cells in posttransplant hematopoietic recovery

Using three different statistical tests in parallel, we showed in a preliminary study that neither mononuclear cells, CD34+ 33+ or 33- cells, nor CD34+ 38+ cells significantly correlated with engraftment kinetics following autologous blood cell transplantation (ABCT). We additionally demonstrated here, in a series of patients suffering from malignant diseases, that the graft content in CD34+ 38- cells is individually a more sensitive indicator of the earliest, as well as the latest post-ABCT trilineage hematopoietic recovery than the colony-forming units-granulocyte-macrophage and even the total CD34+ cell content. This suggests that the CD34+ 38- cell population is itself subdivided into two more subsets, one being already lineage-committed and responsible for short-term engraftment, the other containing only very primitive hematopoietic cells responsible for sustained engraftment. Strong arguments favor the probability that these subsets correspond to HLA-DR+ and DR cells, respectively. We also defined an optimal threshold value of 0.05 x 10(6) CD34+ 38- cells/kg of the patient's body weight (b.w.) above which a rapid and sustained trilineage engraftment safely occurs. In fact, infusion of lower numbers of cells seems to have a more significant impact on long-term compared to short-term neutrophil recovery and on platelet kinetics engraftment. We additionally looked for the eventual influence on engraftment time of the type of disease, and of post-ABCT administration of hematopoietic growth factors (HGF). When the type of disease appeared to have no influence on the engraftment time, posttransplant HGF administration significantly reduced the time to trilineage engraftment in patients transplanted with < 0.05 x 10(6) CD34+ 38- cells, thus justifying it in case of reinfusion of low numbers of CD34+ 38- cells. On the other hand, the administration of HGF after infusion of more than 0.05 x 10(6) CD34+ 38- cells/kg b.w. did not hasten more, or only very little, the engraftment time, thus becoming not only unprofitable for the patients but costly as well.     

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.     

Identification and hematopoietic potential of CD45- clonal cells with very immature phenotype (CD45-CD34-CD38-Lin-) in patients with myelodysplastic syndromes

CD45 is a hematopoietic lineage-restricted antigen that is expressed on all hematopoietic cells except for some mature cell types. Cells expressing CD45 and CD34 but lacking CD38 and lineage antigens (CD45+CD34+CD38-Lin- cells) are well-documented hematopoietic stem cells (HSCs), and CD45+CD34-CD38-Lin- cells are probably less mature HSCs. In myelodysplastic syndromes (MDS), the malignant transformation site is a matter of debate, and CD45+CD34+CD38-Lin- HSCs were recently reported to be clonal. In the study reported here, we detected CD45-CD34-CD38-Lin- cells in the peripheral blood and bone marrow of patients with MDS and isolated them by successive application of density centrifugation, magnetic cell sorting, and fluorescence-activated cell sorting. Fluorescence in situ hybridization showed that CD45-CD34-CD38-Lin- cells had the same chromosomal aberration as the myeloblasts. In addition to CD45- and CD34-, they lacked CD117 and CD133 expression. Generally, MDS cells have extremely reduced hematopoietic potential compared with normal hematopoietic cells, but we documented the following in some patients. Freshly isolated CD45-CD34-CD38-Lin- cells did not form any hematopoietic colonies but had long-term culture-initiating cell activity. When cocultured with stroma cells, CD45-CD34-CD38-Lin- cells showed only weak potential for proliferation and differentiation, yet they differentiated into CD34+ cells and then mature myeloid cells. This newly identified cell population represents the most immature immunophenotype so far identified in the hematopoietic lineage and is involved in the malignant clone in MDS.     

Jaggedl-Notch信号通路在外周CD4+T细胞分化中的作用

病毒的慢性感染与机体的免疫耐受有关.近年来的研究发现,Notch信号通路与机体的免疫系统存在着密切的关系,它从多个方面参与T细胞功能的调控,包括T细胞的活化和增殖、细胞因子的分泌和Th1/Th2分化,也参与调节性T细胞(Treg)的产生、扩增和功能发挥等,表明Notch信号途径不仅参与免疫系统发育,同时在成熟免疫细胞功能调节中也具有重要的作用.