Decreased yield, phenotypic expression and function of immature monocyte-derived dendritic cells in cord blood

Dendritic cells are critical for the induction of both primary immune responses and immunological tolerance, as well as for the regulation of T-helper 1 (Th1) and 2 (Th2) immune responses. As neonates are notably deficient in Th1 response and cord blood transplantation is noted to result in less graft-versus-host disease (GvHD), we compared the phenotypic and functional characteristics of monocyte-derived dendritic cells (DCs) that favour Th1 development from cord blood and adult peripheral blood to understand the underlying mechanisms of these observations. Our results showed that: (1) after culture for 7 d with interleukin (IL)-4 and granulocyte--macrophage colony-stimulating factor (GM-CSF), cord blood monocytes generated less CD1a(+) cells than adult peripheral blood monocytes, and the CD1a+ cell percentage decreased thereafter; (2) compared with adult blood DCs, cord blood DCs had reduced intensity of expression of CD1a and MHC class II molecules, but the expression levels of CD11c and CD86 were similar; (3) the endocytotic ability of cord blood DCs was reduced compared with adult blood DCs, and this function was related to reduced mannose receptor (MR)-positive cells; (4) furthermore, the ability of cord blood DCs to stimulate CD3(+) T cells in an allogeneic mixed lymphocyte reaction was significantly lower than that of adult blood DCs. These results suggested that the dysfunction of cord blood monocytes in differentiating into professional DCs will affect the activation of naive T cells, especially Th1 development, and may be related to the susceptibility to different infections in the neonates, as well as the lower incidence of GvHD in cord blood transplantation.     

Intracellular cytokine production and cytokine receptor interaction of cord mononuclear cells: relevance to cord blood transplantation

A 'cytokine storm' consisting of IL-1, IL-2, IL-12, IFNgamma and TNFalpha is considered important in the development of graft-versus-host disease (GvHD). These cytokines activate effector cells or damage host tissues. Cord blood transplantation has been associated with a low incidence of GvHD. We hypothesized that the low incidence of GvHD relates to the cord mononuclear cells being poor producers of pro-inflammatory cytokines. The cytokine profile (IL-1alpha/beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFNgamma and TNFalpha) of cord blood cells induced by immune stimuli was determined in heparinized whole blood. Compared to adult, cord blood CD3+ and NK cells produced less IFNgamma, less cord blood CD3+ cells and monocytes produced TNFalpha and less monocytes produced IL-1alpha/beta. Although more cord T cells produced IL-2 compared to adult T cells at 4 h, adult T cells produced more at 24 h. Cord blood had similar proportions of monocytes to adult producing IL-6, IL-10 and IL-12. Both adult and cord mononuclear cells constitutively expressed receptors for IFNgamma and TNFalpha but not IL-12. In contrast to the adult cells, cord CD3+ and NK cells did not express IL-12 receptor but did up-regulate IL-10 receptor after mitogenic stimulation. The findings of this study indicate that the cord blood cytokine-receptor network is biased towards anti-inflammatory activity compared to adult and helps to explain the decreased incidence of GVHD in cord blood transplantation.     

Effect of insulin on functional status of cord blood-derived dendritic cells and on dendritic cell-induced CTL cytotoxicity against pancreatic cancer cell lines

BACKGROUND:Dendritic cells(DCs)are the most important antigen-presenting cells in the human body,and DCs with different mature status possess different or even opposite functions.This study was designed to explore the influence of insulin on the functional status of cord bloodderived DCs and on DC-induced cytotoxic T lymphocyte (CTL)activity against pancreatic cancer cell lines. METHODS:Mononuclear cells were isolated from fresh cord blood.Interleukin-4(IL-4)and granulocytemacrophage colony-stimulating fact...     

Phenotypic analysis of functional T-lymphocyte subtypes and natural killer cells in human cord blood: relevance to umbilical cord blood transplantation

Cord blood has been used successfully for stem cell transplantation in several haematological conditions: Fanconi's anaemia, leukaemia and Wiskott-Aldrich syndrome. On account of the low incidence of GVHD observed following cord blood transplantation, it has been suggested that cord blood be used for HLA-matched, or perhaps one or two antigens mismatched, and unrelated stem cell transplantation. Based on an extensive immunophenotype-functional correlation, we determined that cord blood contains mainly immature unprimed T lymphocytes that are predominantly suppressor cells. Recent findings suggest that dysregulated production of cytokines (IL-1, IL-2, TNFα) plays a role in GVHD. We showed that T cells in cord blood express receptors for IL-2, TNFα, but no receptors for IL-1. Similarly, NK cells, one of the effector cells of GVHD, express receptors for TNFα and γIFN but do not express receptors for IL-1, nor IL-2R α-chain (CD25) although IL-2R β-chain is expressed. The potential for activation of T lymphocytes and NK cells therefore exists in the context of bone marrow transplantation. However, the high number of suppressor cells in cord blood most likely modulate the activation of lymphocytes and NK cells thereby minimizing GVHD.     

Human leucocyte antigen-Cw-specific cytotoxic T lymphocytes generated from naive cord blood used for cord blood stem cell transplantation

Human leucocyte antigen (HLA)-Cw-reactive cytotoxic T lymphocytes (CTL) were generated from cord blood (CB) lymphocytes of two cases used for cord blood stem cell transplantation (CBSCT). In both cases, the CTL were cytotoxic against the patient's leukaemic cells, as well as the patient's Epstein-Barr virus (EBV)-lymphoblastoid cell line (EBV-LCL) and phytohaemagglutinin blasts, and the cytotoxicity was blocked by anti-HLA-class I monoclonal antibodies. In the first case, the CTL recognized Cw 3 (Cw 9 and Cw 10)-positive EBV-LCL, while in the second case, the CTL recognized Cw1 and/or Cw7. These cases suggest that CB T cells may be competent enough for generating CTL to induce a graft-versus-leukaemia effect and/or graft-versus-host disease in patients with CBSCT and that the mismatching of Cw antigens between patient and CB may be related to the outcome of CBSCT.     

Improving cord blood transplantation in children

Umbilical cord blood transplantation (UCBT) is widely used to treat children affected by many disorders. In comparison to bone marrow transplantation, the advantages of UCBT are represented by lower incidence and severity of graft- versus -host disease, easier procurement and prompter availability of cord blood cells, and by the possibility of using donors showing human leucocyte antigen disparities with the recipient. Despite these advantages, the large experience gained over the last decade has clearly demonstrated that UCBT patients may be exposed to an increased risk of early fatal complications, due to the lower engraftment rate of donor haematopoiesis, delayed kinetics of neutrophil recovery and lack of adoptive transfer of pathogen-specific memory T-cells. An inverse correlation between the number of nucleated cord blood cells infused per kilogramme recipient body weight and the risk of dying from transplantation-related causes exists. Thus, it is not surprising that strategies aimed at increasing the number of cord blood progenitors, favouring stem cell homing, and transferring pathogen-specific lymphocytes, have been recently investigated. In particular, selection of the richest cord blood units, infusion of 2 units in the same recipient, intrabone injection of cord blood cells, and transplantation of ex-vivo expanded progenitors can contribute to improve the results of UCBT.     

Human dendritic cells express the thrombopoietin receptor, c-Mpl

Human thrombopoietin (TPO) is a haemopoietic growth factor that is essential for the growth and development of megakaryocytes. However, c-Mpl, the TPO receptor, has been detected in human leukaemic cell lines with a myelomonocytic phenotype. These results raise the possibility that dendritic cells (DC), a putative myeloid lineage cell, may also express c-Mpl and respond to TPO. In haemopoietic stem cell transplantation, DC could induce graft-versus-host disease by its strong antigen-presenting capacity. In this study we have examined the effect of TPO on differentiation and the antigen-presenting capacity of DC. To differentiate DC, cord blood CD34+ cells were cultured in the presence of a cytokine cocktail either in serum-free medium or RPMI1640 containing 10% fetal bovine serum. Flow cytometric analysis and immunocytochemical staining demonstrated that c-Mpl was expressed on DC. Furthermore, the expression of c-Mpl mRNA was detected in DC by RNase protection assay. However, when TPO was added to the culture system there were no significant changes in the differentiation and mixed leucocyte-stimulating capacity of DC. These findings suggest that TPO may be administered following cord blood transplantation without significant augmentation of antigen presentation mediated by DC.     

CpG ODN对食管癌细胞RNA转染的脐血树突状细胞诱导CTL抗肿瘤功能的影响

目的探讨食管癌细胞RNA转染脐血树突状细胞（DCs）对细胞毒性T淋巴细胞（CTL）特异性抗肿瘤作用的影响,以及CpG寡脱氧核糖核苷酸（CpG ODN）的免疫佐剂作用。方法分离脐血单个核细胞,经SCF、GM-CSF和IL-4诱导和食管癌RNA转染形成成熟DCs,加入CpG ODN,检测DCs表面标志及DCs诱导的T细胞增殖、CTL杀伤活性。结果食管癌RNA转染后DCs高表达抗原提呈分子、协同刺激分子和黏附分子,对T细胞的促增殖作用和CTL杀伤活性显著增强（P〈0.01）,CpG ODN能显著促进DCs功能。结论CpG ODN具有增强食管癌细胞RNA转染的DCs对T细胞的促增殖作用和促CTL杀伤活性的作用。     

Kinetics of regulatory dendritic cells in inflammatory responses during Trypanosoma evansi infection

Trypanosoma evansi (T. evansi) causes a wasting disease in almost all mammals. Trypanosoma evansi infection gives rise to the inflammatory responses that contribute to the development of inflammation‐associated tissue injury. To determine what kinds of inflammatory molecules play roles in the pathogenicity of T. evansi infection, polymerase chain reaction array analysis was performed on samples from the infected and uninfected mice. The inflammatory cytokine and chemokine storm, caused mainly by macrophages, was observed. On the other hand, the expression levels of Ccl8 and Il10 in splenocytes were also markedly increased. These results suggested an augmentation in the number and activity of regulatory dendritic cells (DCs). Therefore, the kinetics of regulatory DCs in T. evansi‐infected mice were investigated. During T. evansi infection, the regulatory DCs became prevalent, with reducing the amount of inflammatory DCs. Interestingly, when the regulatory DCs were implanted into T. evansi‐infected mice, the survival was prolonged, and the expression levels of inflammatory molecules were suppressed. Taken together, these results showed that a subset of regulatory DCs acted as a potential regulator of the inflammatory responses.     

Donor cell derived acute myeloid leukemia after allogeneic cord blood transplantation in a patient with adult T-cell lymphoma

We report a patient with adult T-cell lymphoma who developed acute myeloid leukemia (AML) after allogeneic cord blood transplantation (CBT). Fluorescence in situ hybridization (FISH) studies and molecular analysis using short tandem repeat (STR) sequences proved the AML to be of donor origin. Although 25 cases of donor cell leukemia (DCL) occurring after allogeneic bone marrow transplantation have previously been reported, there have been no reports of DCL after CBT. This case is the first-reported DCL patient after CBT.     

细胞因子对脐血树突状细胞体外诱导分化及扩增作用的研究

目的:树突状细胞是初始免疫反应中最重要的抗原提呈细胞。本研究目的是分析脐带血的细胞组成,研究加入细胞因子培养前后脐血树突状细胞的变化,以脐血为来源探索体外诱导、扩增树突状细胞的方法并进行表型鉴定。方法:脐血12例,分离单个核细胞。在脐血单个核细胞中加入细胞因子粒-巨噬细胞集落刺激因子(GM-CSF)、白细胞介素-3(IL-3)、造血干细胞生长因子(SCF)和促红细胞生成素(EPO),培养4周。应用流式细胞仪,使用CD4、CD8、CD19、CD34、CD38、CD1a、CD11c和CDw123单克隆抗体,测定培养前及培养后1、2、3、4周脐血细胞的细胞表面抗原变化及树突状细胞情况。结果:新鲜脐血单个核细胞中CD34 造血干细胞0.22×108/L、CD1a 细胞0.27×108/L、CD11c 细胞5.87×108/L、CD83 细胞1.94×108/L、CDw123 细胞2.73×108/L。加入细胞因子GM-CSF、IL-3、SCF、EPO后培养1~4周的脐血单个核细胞分化为CD1a 、CD11c 、CD83 、CDw123 树突状细胞,在培养的2~4周,脐血树突状细胞数量明显增多,此后逐渐减少。通过培养,树突状细胞数量增加达CD1a 细胞11.02×108/L、CD11c 细胞28.24×108/L、CD83 细胞10.57×108/L、CDw123 细胞18.7×108/L。结论:在培养液中加入细胞因子GM-CSF、IL-3、SCF和EPO后培养2~4周,脐血单个核细胞可分化为CD1a 、CD11c 、CD83 、CDw123 树突状细胞。这些树突状细胞作为抗原提呈细胞在肿瘤免疫治疗上将起到重要作用。     

Interleukin 10, produced in abundance by human newborn T cells, may be the regulator of increased tolerance associated with cord blood stem cell transplantation

Summary. The use of human umbilical cord blood as an alternative source of stem cells to bone marrow for the reconstitution of the immune system is associated with less frequent and less severe incidence of graft‐versus‐host disease (GVHD). This study focuses on aspects of cord blood T‐cell biology that may contribute to a perceived increased tolerance associated with the neonatal immune response. A skewing of the T‐helper (Th)1/Th2 phenotype of cord blood T cells towards a Th2 response has frequently been cited as a possible cause. In this study, primary and repeated stimulation via the T‐cell receptor (TCR) complex induced a Th0‐type cytokine response, with both adult and cord blood‐derived naïve T cells producing interferon γ (IFN‐γ), interleukin 4 (IL‐4) and IL‐5. IL‐10 was induced in cord blood T‐cell cultures during primary stimulation, while adult T cells began to secrete IL‐10 only after repeated stimulation. The presence of the antigen‐presenting cell (APC)‐derived cytokine IL‐1β inhibited IL‐10 production by cord blood cells. The effects of IL‐12 and IL‐4 on T‐cell cytokine responses were also examined. In addition to their differential Th1/Th2 skewing effects on cord and adult T cells, both cytokines augmented IL‐10 production in both T‐cell populations. These findings demonstrate that cord blood T cells may secrete large amounts of the anti‐inflammatory cytokine IL‐10 and that the presence of IL‐1β or Th1/Th2 skewing cytokines can regulate its production. This data provides support for the recognized tolerant nature of the newborn immune response that may contribute to the reduced incidence of GVHD associated with cord blood transplantation.     

Indirect evidence that maternal microchimerism in cord blood mediates a graft-versus-leukemia effect in cord blood transplantation

During pregnancy women can develop B- and T-cell immunity against the inherited paternal antigens (IPAs) of the fetus, such as HLA, peptides of minor histocompatibilty antigens, and possibly onco-fetal antigens. The biological and pathological role of these pregnancy-induced immunological events is only understood in part. However, anti-IPA immunity in the mother persists for many decades after delivery and may reduce relapse in offspring with leukemia after HLA-haploidentical transplantation of maternal hematopoietic stem cells (HSC). We hypothesized that maternal anti-IPA immune elements cross the placenta and might confer a potent graft-versus-leukemia effect when cord blood (CB) is used in unrelated HSC transplantation. In a retrospective study of single-unit CB recipients with all grafts provided by the New York Blood Center, we show that patients with acute myeloid or lymphoblastic leukemia (n = 845) who shared one or more HLA-A, -B, or -DRB1 antigens with their CB donor's IPAs had a significant decrease in leukemic relapse posttransplantation [hazard ratio (HR) = 0.38, P < 0.001] compared with those that did not. Remarkably, relapse reduction in patients receiving CB with one HLA mismatch (HR = 0.15, P < 0.001) was not associated with an increased risk of severe acute graft-versus-host disease (HR = 1.43, P = 0.730). Our findings may explain the unexpected low relapse rate after CB transplantation, open new avenues in the study of leukemic relapse after HSC transplantation (possibly of malignancies in general), and have practical implications for CB unit selection.     

Infectious complications following unrelated cord blood transplantation

INTRODUCTION: Infections following cord blood transplantation are just beginning to be defined in the literature. This review will outline infections at death, the epidemiology of individual infections, and the impact of stem cell source. METHODS: A review of studies published since 2000. RESULTS: Based on registry data, most studies demonstrate an approximate rate of infection at death of 30-40% among cord blood recipients. Bacterial infections often occur prior to engraftment and increase among patients with graft failure. In addition, there is delayed recovery of the immune response among patients with graft-versus-host disease that leads to viral infections at later time points. The risk of serious infection among children receiving umbilical cord blood (UCB) grafts is comparable to that of children receiving unmanipulated marrow and is lower than that of recipients of a T-cell-depleted stem cell source. Among adult patients, despite an overall higher incidence of serious infections after UCB transplantation as compared with unrelated donor grafts, non-relapse mortality and overall survival were not significantly different between haematopoietic stem cell sources. CONCLUSIONS: Further studies are needed to confirm these observations and determine whether the risk of infection for cord blood recipients is comparable to that of recipients of unmanipulated marrow.     

Quantification of progenitors capable of generating T cells in human cord blood

Objective:  For transplantation of cord blood (CB) cells, it is important to select a CB sample that can reconstitute not only myelo-erythropoiesis but also lymphopoiesis in recipients. However, until now the reconstitution ability of CB samples has been assessed by colony forming unit-culture (CFU-C) assay or by simply counting CD34⁺ cells. The present study aims at establishing a method capable of assessing the potential of T lymphopoieses of CB samples.
Methods:  CD34⁺CD38⁻ cells sorted from CB were cultured on a monolayer of murine stromal cell line TSt-4, transduced with the human Delta-like 1 gene.
Results:  Immature T cells expressing CD5 and/or CD7 were generated in the culture. As these immature T cells can easily be discriminated from mature T cells that are included in the mononuclear cell population (MNCs), we can use the MNCs as starting material for quantification of progenitors capable of generating T cells (TGP). By applying a limiting dilution analysis, we succeeded in determining the frequency of TGP in MNCs. It was found that the ratios for the number of TGP vs. that of CFU-C differ among CB samples maximally by 3.5 times.
Conclusion:  The present assay system provides a novel tool for the evaluation of CB samples, especially for their T-cell-generating potential.     

脐带血干细胞治疗失代偿期原发性胆汁性肝硬化疗效观察

目的观察脐带血干细胞治疗失代偿期原发性胆汁性肝硬化患者的安全性及对肝功能的改善作用。方法选择失代偿期原发性胆汁性肝硬化患者60例,随机分为治疗组30 例和对照组30例。对照组行护肝治疗,治疗组在此基础上经股动脉插管至肝固有动脉,注入脐带血干细胞治疗。结果治疗后8 w,治疗组和对照组血清ALB 水平分别为（33.1±8.3）g/L 和（25.6±7.6）g/L,PTA 分别为（43.0±11.4）%和（38.2±12.5）%,差异有显著性（P<0.05） ;GGT水平分别为（250.3±17.5）U/L 和（360.3±16.5）U/L,ALP水平分别为（380.3±16.5）U/L 和（510.3±17.5）U/L,IgM水平分别为（21.3±16.5）g/L 和（32.5±18.5）g/L,具有显著性差异（P<0.05）。结论脐带血干细胞治疗失代偿期原发性胆汁性肝硬化可以改善肝脏的合成、代谢功能,调节免疫功能,安全性好。     

氧化苦参碱对慢性HBV携带者外周血树突状细胞及细胞因子的影响

目的：研究氧化苦参碱对慢性乙型肝炎病毒（ HBV ）携带者外周血树突状细胞及细胞因子的影响。方法：分离60例慢性HBV携带者外周血单核细胞后在诱导培养树突状细胞过程中治疗组加入氧化苦参碱,对照组不加,每组各30例,然后提取两组成熟的树突状细胞,计数后采用双标记流式细胞分析技术检测其活性,再用表位多肽刺激后,和自体淋巴细胞共育,分别检测IL-2和IFN-γ（ Th1分泌）及IL-4和IL-10（ Th2分泌）。结果：治疗组其树突状细胞数目增多,共刺激分子CD1a、 CD80、 CD86明显高于对照组（P＜0.05）。与自体淋巴细胞共育后产生的IL-2、 IFN-γ明显高于对照组（P＜0.05）; IL-4、 IL-10与对照组差异无显著性意义（P＞0.05）。结论：氧化苦参碱可增多、增强HBV携带者外周血树突状细胞的活性,当其与自体淋巴细胞共育时,可增强Th1类细胞因子的表达,对Th2类细胞因子无明显影响。     

Effect of ex vivo cytokine treatment on human cord blood engraftment in NOD-scid mice

Umbilical cord blood transplantation is considered an alternative to traditional bone marrow transplantation for patients who do not have matched sibling donors. In this study, we examined the effects of ex vivo treatment of human cord blood cells with cytokine mixtures and assessed the ability of treated cells to engraft in NOD-scid mice. We incubated the cord blood with a four-factor cytokine mixture of interleukin (IL)-3, IL-6, IL-11 and stem cell factor, or with a two-factor cytokine mixture of thrombopoietin and flt-3. Incubation of cord blood for 48 h with either cytokine mixture did not affect progenitor cell number or proliferative potential as measured by the high proliferative potential (HPP) assay. Cytokine-treated cord blood injected into irradiated NOD-scid mice resulted in multilineage human engraftment. Overall, incubation with cytokines resulted in variable levels of engraftment with different cord blood samples. Incubation of cord blood with the four-factor cytokine mixture resulted in increased survival of irradiated NOD-scid recipients. These results demonstrate that short-term ex vivo treatment of human progenitor cells gives variable results on in vivo multipotential capabilities.     

Impact of obstetric factors on cord blood donation for transplantation

Recent reports have shown that low nucleated cell dose significantly decreases survival after cord blood transplantation. Prior to starting clinical cord blood banking we investigated the impact of obstetric factors on cell dose and volume of cord blood donations. Cord blood was obtained from 114 normal full-term deliveries. Mean volume collected was 93.5 ml, mean total nucleated cell count (TNC) was 13.1 x 108. Statistical analysis was by backwards stepwise regression. Significant factors affecting nucleated cell yield were volume of blood collected (P < 0.001), length of gestation (P < 0. 0001), time from delivery of the infant to cord clamping (P = 0.018) and total length of labour (P = 0.002). In clinical cord blood banking we have successfully used these findings for pre-collection assessment of placentae. Out of 476 cord blood donations subsequently collected for banking, only 29 (6.1%) have been discarded due to low volume. The mean TNC of the 409 banked units following volume reduction was 10.1 x 108. Despite careful optimization of collection, processing and storage techniques, cell dose still limits cord blood transplantation to smaller recipients.