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.     

The preTCR-dependent DN3 to DP transition requires Notch signaling, is improved by CXCL12 signaling and is inhibited by IL-7 signaling

The requirement for Notch signaling during T-cell development has been extensively studied. Nevertheless, the developmental stage at which it is required and whether additional signaling pathways are needed are still poorly understood. By using a stromal-cell-free culture system, we show that sorted double-negative 3 (DN3) thymocytes only require a Delta-like-4-induced Notch signal to differentiate into double-positive (DP) cells. This differentiation process is preTCR-α dependent. DN3 cells undergo 4-5 proliferation cycles, and the addition of the chemokine CXCL12 improves proliferation. IL-7 blocks the differentiation of DN3 cells to DP cells but not the Notch-induced proliferation of cultured DN3 cells. The impaired differentiation correlates with an inhibition of Rag-2 up-regulation. Overall, the in vitro stromal-cell-free culture system presented here also provides a powerful and unique tool for studying the mechanisms involved in the positive and negative selection of T cells.     

Notch信号调节外周T细胞的活化、增殖与分化

初始T细胞分化为效应T和记忆T细胞受到多种因素调节.最近在Notch信号途径的研究进展显示它也参于T细胞的活化与分化.大量研究已经表明Notch信号途径可以影响T细胞在中枢免疫器官的发育,现在关于它调节外周T细胞的分化状态也积累不少证据,Notch信号活化之后能够改变CD4+和CD8+T细胞分泌细胞因子的特点.以下着重介绍Notch信号参于调节外周T细胞的活化、增殖和分化的最新资料,尽管不同的研究者所得实验结果有冲突之处,但已经提示Notch信号在T细胞外周发育中的重要意义,特别重要的是抗原递呈细胞(APC)可以通过Notch信号途径调节T细胞的分化.     

Notch signaling in differentiation and function of dendritic cells

Hematopoietic stem cells give rise to multiple lineages of cells. This process is governed by a tightly controlled signaling network regulated by cytokines and a direct cell-cell contact. Notch signaling represents one of the major pathways activated during direct interaction between hematopoietic progenitor cells and bone marrow stroma. A critical role of Notch signaling in differentiation of T- and B-lymphocytes has now been established. Until recently, the role of Notch signaling in the development of myeloid cells and particular dendritic cells remained unclear. In this review, we discuss recent exciting findings that shed light on the critical role of Notch in differentiation and the function of dendritic cells and its impact on immune responses.     

Pre-TCR signaling regulates IL-7 receptor alpha expression promoting thymocyte survival at the transition from the double-negative to double-positive stage

The pre-T cell receptor (pre-TCR) and IL-7 receptor (IL-7R) are critical mediators of survival, proliferation and differentiation in immature thymocytes. Here we show that pre-TCR signaling directly maintains IL-7Ralpha expression as developing thymocytes undergo beta-selection. Inhibition of IL-7/IL-7R signaling in (CD44-CD25-) DN4 cells results in decreased generation of double-positive thymocytes due to increased death of rapidly proliferating beta-selected cells. Thus, we identify a mechanism by which pre-TCR signaling controls the selective survival of TCRbeta+ thymocytes, and define a further stage of T cell differentiation in which signaling from a TCR regulates the ability of that cell to respond to cytokine.     

Non-redundant role for IL-7R signaling for the survival of CD8+ memory T cells

IL-7 and IL-15 are important cytokines for CD8 memory T cells. However, the extent that IL-7 is essential for CD8 T cell memory remains unclear because blocking IL-7 in vivo results in near complete inhibition of T cell development with the few mature T cells exhibiting functional abnormalities. To bypass this complication, CD8 memory development was examined utilizing a mouse model where transgenic IL-7Ralpha was selectively expressed in the thymus of IL-7Ralpha(-/-) mice. T cell development was corrected but the resulting peripheral T cells were essentially IL-7 non-responsive. Activation of IL-7R-defective OT-I CD8(+) T cells with OVA(257-264) and IL-2 readily yielded CTL. Upon further culture with IL-15, these CTL expressed phenotypic and functional properties of central memory-like cells. Thus, IL-7R-defective CD8(+) T cells do not exhibit intrinsic defects in effector or memory development. When IL-7R-defective OT-I CTL were adoptively transferred into normal or IL-15(-/-) recipient mice in a non-inflammatory setting, they converted into memory-like cells, but did not persist, which was even more striking in IL-15(-/-) recipients. This poor persistence was rescued after expression of transgenic Bcl-2 in IL-7R-defective OT-I T cells. Collectively, these data indicate that IL-7 is non-redundantly required for the survival of CD8 memory T cells.     

Regulation of endothelial cell differentiation and arterial specification by VEGF and Notch signaling

Analysis of molecular and cellular mechanisms underlying vascular development in vertebrates indicates that initially vasculogenesis occurs when a primary capillary plexus forms de novo from endothelial cell precursors derived from nascent mesodermal cells. Transplantation experiments in avian embryos demonstrate that embryonic endothelial cells originate from two different mesodermal lineages: splanchnic mesoderm and somites. Genetic analysis of mouse and zebrafish reveals that vascular endothelial growth factor (VEGF)/Flk1 and Notch signaling play crucial roles throughout embryonic vascular development. VEGFA plays a major role in endothelial cell proliferation, migration, survival, and regulation of vascular permeability. Flk1, the primary VEGFA receptor, is the earliest marker of the developing endothelial lineage and is essential for endothelial differentiation during vasculogenesis. Notch signaling has been demonstrated to directly induce arterial endothelial differentiation. Recent studies suggest that Notch signaling is activated downstream of VEGF signaling and negatively regulates VEGF-induced angiogenesis and suppresses aberrant vascular branching morphogenesis. In addition to altering endothelial cell fate through Notch activation, VEGFA directly guides endothelial cell migration in an isoform-dependent manner, modifying vascular patterns. Interestingly, genetic studies in mice show that many molecules involved in VEGF or Notch signaling must be tightly regulated for proper vascular formation. Taken together, VEGF and Notch signaling apparently coordinate vascular patterning by regulating each other.     

Toll-like receptor 4-mediated signaling regulates IL-7-driven proliferation and differentiation of B-cell precursors

Lipopolysaccharide （LPS） is known to be a potent activator of mature B cells by signaling through Toll-like receptor 4 （TLR4）. Its impact on early B-cell development, however, is not well defined. When comparing to C3H/HeN mice, TLR4-mutant C3H/HeJ mice showed an increase in the number of pro-B and pre-B cells in the bone marrow. When cultured in the presence of IL-7, the proliferation of pro-B and large pre-B cells was significantly inhibited by LPS, possibly due to reduced IL-7 receptor-a （IL-7Ra） expression. Meanwhile, the generation of IgM＋/IgD＋ B cells was greatly enhanced in IL-7 cultures of pro-B and pre-B cells. Consistent with these results, treatment with LPS facilitated the progression of adoptively transferred B220＋IgM-IgD- precursors into IgD＋ cells. Overall, these data suggest that LPS has a profound influence on early B-cell development, which may contribute to the deregulated B-cell development under physiological and pathological conditions such as bacterial infections.     

Notch信号通路及其在肺癌发生中的作用

Notch信号通路存在于多种动物体内,是许多细胞信号转导通路的交汇点,不仅对正常组织、细胞的分化、发育起重要作用,而且和一些肿瘤的发生、发展相关。Notch信号在肺癌中的作用多样，在不同类型肺癌中呈现出不同的促癌或抑癌功能。了解Notch和肺癌的关系有利于进一步阐明肺癌发生机制,提出预防和治疗肺癌的新途径，为肿瘤基因治疗提供一个新的有希望的靶点。     

IL-7对PHA-抗CD3 LAK细胞抑制效应的研究

文章将IL 7与IL 2 ,PHA及αCD3联用诱生PHA αCD3LAK细胞 ,应用活细胞计数、MTT和APAAP等方法测定了细胞增殖活性、mIL 2R的表达、外源性IL 2的消耗、细胞毒活性和效应细胞表型等指标 ,并分析比较了IL 7的加入对以上指标的影响。结果表明IL 7的加入使PHA αCD3LAK细胞消耗IL 2的量、mIL 2R表达的量和细胞的增殖能力降低 ,细胞毒活性减弱 ,并伴有CD8+ 细胞百分率的下降。这一结果呈现出的IL 7的抑制作用尚未见报道 ,有进一步研究的价值。     

Deficient CCR7 signaling promotes TH2 polarization and B-cell activation in vivo

The chemokine receptor CCR7 has a central role in regulating homing and positioning of T cells and DCs to lymph nodes (LNs) and participates in T‐cell development and activation. In this study, we addressed the role of CCR7 signaling in T_H2 polarization and B‐cell activation. We provide evidence that the lack of CCR7 drives the capacity of naïve CD4⁺ T cells to polarize toward T_H2 cells. This propensity contributes to a lymph node environment in CCR7‐deficent mice characterized by increased expression of IL‐4 and increased frequency of T_H2 cells. We show that elevated IL‐4 levels lead to B‐cell activation characterized by up‐regulated expression of MHC class II, CD23 and CD86. Activated B cells are in turn highly efficient in presenting antigen to CD4⁺ T cells and thus potentially contribute to the T_H2 microenvironment. Taken together, our results support the idea of a CCR7‐dependent patterning of T_H2 responses, with absent CCR7 signaling favoring T_H2 polarization, dislocation of T helper cells into the B‐cell follicles and, as a consequence, B‐cell activation.     

Endothelial cells promote neural stem cell proliferation and differentiation associated with VEGF activated Notch and Pten signaling

To investigate whether and how endothelial cells affect neurogenesis, we established a system to co‐culture endothelial cells and brain slices of neonatal rat and observed how subventricular zone cells differentiate in the presence of endothelial cells. In the presence of endothelial cells, neural stem cells increased in number, as did differentiated neurons and glia. The augmentation of neurogenesis was reversed by diminishing vascular endothelial growth factor (VEGF) expression in endothelial cells with RNA interference (RNAi). Microarray analysis indicated that expression levels of 112 genes were significantly altered by co‐culture and that expression of 81 of the 112 genes recovered to normal levels following RNAi of VEGF in endothelial cells. Pathway mapping showed an enrichment of genes in the Notch and Pten pathways. These data indicate that endothelial cells promote neural stem cell proliferation and differentiation associated with VEGF, possibly by activating the Notch and Pten pathways. Developmental Dynamics 239:2345–2353, 2010. © 2010 Wiley‐Liss, Inc.     

IL-2Rbeta links IL-2R signaling with Foxp3 expression

Immunological tolerance to self antigens is a tightly regulated process. Recent work has demonstrated that the forkhead family member Foxp3 is a critical element in the differentiation and function of mouse CD4(+)CD25(+) regulatory T cells (Treg). Recent work has suggested an important role for IL-2 in the development and maintenance of Treg. To directly assess the effect of IL-2 signaling on Treg development and function, we analyzed mice that were genetically deficient in components of the IL-2 receptor (IL-2R). Mice lacking CD25 (IL-2Ralpha) displayed a slight decrease in Treg within the thymus, while peripheral numbers are unchanged. In contrast, we found that mice deficient in CD122 (IL-2Rbeta) had a profound reduction in both thymic and peripheral Treg, coinciding with more rapid development of a fatal lymphoproliferative disease. Expression of a Foxp3 transgene restored Treg and protected against the onset of autoimmunity. Thus, a signal mediated by IL-2Rbeta is essential for the development and homeostasis of Foxp3(+) Treg in vivo.     

骨髓间充质干细胞体外增殖及分化过程中Notch信号的表达变化

目的研究Notch信号系统对骨髓间充质干细胞的增殖与分化的调控作用。方法用密度梯度离心法分离培养犬骨髓间充质干细胞,观察其增殖及传代情况,并进行成骨、成软骨及成心肌细胞诱导,Western blot检测细胞增殖及分化过程中Notch信号蛋白的表达。结果骨髓间充质干细胞呈梭形、旋涡样生长,增殖及传代能力强,可在体外形成钙结节,分化为软骨细胞,免疫组化示心肌细胞标志物的表达。Western blot显示在增殖过程中可见Notch信号蛋白的表达,为1.00±0.13,当分化为骨细胞及心肌细胞时,其表达上调,分别为1.20±0.14及1.70±0.17,与增殖的干细胞相比有明显差异(P<0.05)。结论 Notch信号系统对骨髓间充质干细胞的增殖及分化起重要的调控作用。     

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.     

NK cells modulate the cytotoxic activity generated by Mycobacterium leprae-hsp65 in leprosy patients: role of IL-18 and IL-13

Protection against intracellular pathogens such as Mycobacterium leprae is critically dependent on the function of NK cells at early stages of the immune response and on Th1 cells at later stages. In the present report we evaluated the role of IL-18 and IL-13, two cytokines that can influence NK cell activity, in the generation of M. leprae-derived hsp65-cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) of leprosy patients. We demonstrated that IL-18 modulates hsp65-induced CTL generation and collaborates with IL-12 for this effect. In paucibacillary (PB) patients and normal controls (N) depletion of NK cells reduces the cytolytic activity. Under these conditions, IL-12 cannot up-regulate this CTL generation, while, in contrast, IL-18 increases the cytotoxic activity both in the presence or absence of NK cells. IL-13 down-regulates the hsp65-induced CTL generation and counteracts the positive effect of IL-18. The negative effect of IL-13 is observed in the early stages of the response, suggesting that this cytokine affects IFNgamma production by NK cells. mRNA coding for IFNgamma is induced by IL-18 and reduced in the presence of IL-13, when PBMC from N or PB patients are stimulated with hsp65. Neutralization of IL-13 in PBMC from multibacillary (MB) leprosy patients induces the production of IFNgamma protein by lymphocytes. A modulatory role on the generation of hsp65 induced CTL is demonstrated for IL-18 and IL-13 and this effect takes place through the production of IFNgamma.