Human Cord Blood Suppressor T Lymphocytes: I. Phenotype and Target of the Inducer of Suppressor Cell Factor

ABSTRACT: The present study examines the characteristics of the cord suppressor T cells and their targets. When an inducer of suppressor T cell factor (TisF) from culture supernatant of alloantigen-activated cord lymphocytes was used as a source of suppression, adult lymphocyte response to soluble antigen candidin, alloantigens, and pokeweed mitogen were all suppressed. These results suggest that TisF acts primarily on T cells in an antigen-nonspecific fashion. Furthermore, pre-culture T cell irradiation, but not T8⁺ cell depletion, of adult lymphocytes abrogated the suppressive effect of TisF on the PWM-stimulated IgG production, suggesting that the target of TisF belongs to the radiosensitive T8^?(T4⁺) T cell subset. When different cord T cell subsets were tested for suppressor activity, only radiosensitive T4⁺ cells were capable of suppressing IgG production by adult lymphocytes. Neither preculture T8⁺ cell depletion nor irradiation of adult T cells affected the suppression. Taken together, these results suggest that the suppressor activity of cord T cells originated from an inducer of suppressor ceils that activates radiosensitive T4⁺ suppressor effector cells through TisF. In turn, these effector cells act on either radioresistant T4⁺ helper T cells or B cells or both.     

Programmed Cell Death (Apoptosis) in Cord Blood Lymphocytes

Cord blood lymphocytes are functionally immature and have deficient immune responses. In order to determine whether the process of programmed cell death is distinct between cord blood and peripheral blood lymphocytes, we analyzed the expression of fas and bax (apoptosis promoting genes) and bcl-2 and bcl-x _L (apoptosis inhibiting genes) at protein or mRNA levels using flow cytometry and quantitative PCR methods, respectively. The susceptibility of T cell subsets from cord blood and adult peripheral blood to undergo apoptosis following restimulation with anti-CD3 or anti-Fas monoclonal antibodies was also studied. We observed that cord blood T cell subsets expressed lower levels of Fas and Bcl-2, a low bcl-2/bax ratio, and higher bcl-x _L compared to peripheral blood. Additionally, upon primary stimulation with anti-CD3, cord blood T cell subsets were more resistant to apoptosis compared to peripheral blood. In contrast, rechallenge of previously stimulated lymphocytes with anti-CD3 monoclonal antibody triggered apoptosis in a larger proportion of T cells from cord blood as compared to peripheral blood, whereas the number of T cells undergoing anti-Fas-induced programmed cell death were lower in cord blood compared to peripheral blood.     

脐血红细胞免疫功能及流变特性的变化

目的 :研究脐血 4℃保存 3周红细胞免疫功能及流变特性的变化。方法 :取正常孕妇脐血 36份 ,无菌采血、4℃保存。在采血后 2h、1周末、2周末、3周末应用红细胞C3b受体花环试验和红细胞免疫复合物花环试验检测红细胞C3b受体活性 (RBC C3b)和红细胞粘附免疫复合物 (IC)率 ,同时应用LBY N6A旋转式粘度仪检测红细胞粘度、红细胞聚集指数 (AI)、刚性指数 (IR)及红细胞压积。结果 :脐血保存 2周内RBC C3b、IC、高切粘度和IR无显著性变化 ,3周末差异显著 ;AI则在 2周末显著上升 ,第 3周末上升更加明显 ;红细胞压积 3周内无显著变化。结论 :随着脐血保存时间的延长 ,红细胞的免疫功能在逐渐下降 ,同时对红细胞的变形性及红细胞的聚集性会产生重要影响 ,特别是第 3周出现显著性变化     

从人外周血B淋巴细胞中应用PCR扩增人抗体基因

本文用常规ＰＣＲ法和半套式ＰＣＲ法，以一组人抗体重链和轻链引物，直接从人外周血淋巴细胞中扩增出抗体重链Ｆｄ基因和轻链基因。一些常规ＰＣＲ法不能直接扩增的人抗体基因，用半套式ＰＣＲ法扩增却得到了阳性结果。扩增的抗体基因的分子量与国内外同类报道一致。本文结果提示，在建立抗体基因文库时，半套式ＰＣＲ法能进一步丰富扩增的抗体基因的多样性     

细胞因子对脐血NK细胞分化及调节活性的作用

脐血来源丰富和GVHD发生率低等特点使脐血越来越多地用于造血干细胞移植,但因脐血淋巴细胞免疫活性较低,移植后GVL相对较弱,因而不利于长期生存。已有研究发现NK细胞可杀伤宿主抗原呈递细胞而减轻GVHD,也杀伤肿瘤细胞起GVL作用,因此,如何提高脐血NK细胞活性已成为脐血造血干细胞中热门课题。近年发现一些细胞因子具有促进NK细胞分化成熟及增强抗肿瘤作用,因此细胞因子有望用于临床,在脐血移植中起重要作用。     

脐带血中衍生树突状细胞及其生物学特性的研究

目的　利用脐带血衍生培养树突状细胞 (DC) ,以获得可用于临床治疗的一种新的血细胞制品 .方法　从脐带血中分离单个核细胞 ,联合使用细胞因子GM -CSF、IL - 4和TNF -α在体外诱导和扩增DC ;采用扫描电镜、流式细胞仪 (FACS)和混合淋巴细胞反应 (MLR)试验对脐带血来源的DC进行生物学特性分析 .结果　从 30ml脐血诱导培养 12d后可获得 6 .6× 10 6的DC .扫描电镜观察具有典型的树突状突起 ;FACS分析显示 ,获得的DC细胞群高表达CD1a(90 .6 % )、CD80 (96 .8% )、CD86(84 .8% )和HLA -DR(91.0 % ) ;MLR显示 ,脐带血诱导培养的DC对同种异体的脐带血幼稚型T细胞均具有强烈的激发和促增殖作用 .结论　从脐带血中成功诱导出大量高纯度成熟DC ,为依赖DC免疫治疗的临床应用打下基础     

脐血血管内皮祖细胞的分离和诱导分化

目的 从脐血中分离内皮祖细胞，诱导其向内皮细胞分化，研究内皮祖细胞的生物学特性和诱导分化条件。方法 从新鲜脐血中纯化的CD133^ 细胞接种于添加了VEGF、bFGF、IGF—1的M199培养液中，观察梭形贴壁细胞的出现时间和特异性细胞标志。结果 培养3—4d可观察到梭形贴壁细胞，14d左右可形成索条状结构，贴壁细胞表达血管内皮细胞特异性标志VE-cadherin,vWF,UEA-1和VEGFR-2。结论 脐血中含有内皮祖细胞，在一定的条件下，可分化为内皮样细胞。     

The In Vitro Effect of a Calf Thymus Extract (Thymostimulin) on T Cell Phenotypes in Cord Blood Lymphocytes

ABSTRACT: An investigation was carried out on the in vitro effect of a calf thymus extract, thymostimulin, on the distribution of T cell phenotypes as defined by OKT3, OKT4, and OKT8 murine monoclonal antibodies and on E-rosetting cells in human cord blood lymphocytes from healthy newborns. The percentages of E-rosette-forming lymphocytes and OKT3⁺ total T population were lower in newborns than in adults (E-rosettes: 43.8% ± 13% vs 57.9% ± 7.9%, p < 0.01; OKT3⁺ cells: 53.3% ± 15.5% vs 79.9% ± 4.7%, p < 0.01), while the OKT4⁺/OKT8⁺ (helper/suppressor) cell ratio was normal in both (newborns: 3.40; adults: 2.44—NS). Thymostimulin increased the number of E-rosette-forming cells from 43.8% ± 13% to 49.9% ± 12.7% (p < 0.01), as well as the percentage of phenotypic T lymphocytes. The highest increases were observed in the OKT4⁺ cells (37.7% ± 14% to 49.1% ± 13.8%, p < 0.001), while smaller changes were observed in the OKT3⁺ cells (53.3% ± 15.5% to 58.1% ± 13.3%, p < 0.02) and OKT8⁺ cells (12.8% ± 6.4% to 16.6% ± 6.5%, p < 0.02). The results of the present study suggest that thymostimulin mainly provokes an increase in the helper T cell phenotype in cord blood lymphocytes.     

外周血B淋巴细胞半套式PCR法扩增人抗体基因

目的用半套式PCR法，以一组人抗体重链和轻链引物直接从人外周血淋巴细胞中扩增人全套抗体基因片段．方法从不同人群外周血淋巴细胞中提取总RNA，经反转录后，以免疫球蛋白信号肽序列引物和家族特异性免疫球蛋白可变区基因引物，进行半套式PCR扩增人全套抗体基因片段．结果采用不同的引物进行重链、轻链Kappa和Lambda链的半套式PCR扩增，均能获得相应大小的PCR产物，其结果扩增率达到100％．结论在建立抗体基因文库时，半套式PCR法能进一步丰富扩增的抗体基因的多样性，可弥补由于转化效率不高而降低抗体库多样性的不足．     

Cord Blood Stem Cells: A Review of Potential Neurological Applications

It is estimated that as many as 128M individuals in the United States, or 1 in 3 people, might benefit from regenerative medicine therapy. Many of these usages include applications that affect the nervous system, including cerebral palsy, stroke, spinal cord injury and neurodegenerative disease such as Parkinson’s. The numbers of such individuals affected range from 10,000 (for cerebral palsy) to 700,000 annually (for stroke) at a cost of more than $65B. For the foreseeable future, regenerative medicine entrée to the clinic will depend upon the development of adult or non-embryonic stem (ES) cell therapies. Currently, non-ES cells easily available in large numbers from affected individuals can be found in the bone marrow, adipose tissue and umbilical cord blood (CB). It is our belief that CB stem cells are the best alternative to ES cells as these stem cells can be used to derive tissues from the mesodermal, endodermal and ectodermal germ lineages. CB contains a mixture of different types of stem cells in numbers not seen in any other location including embryonic-like stem cells, hematopoietic stem cells, endothelial stem cells, epithelial stem cells, mesenchymal stem cells and unrestricted somatic stem cells. This review will summarize the findings reported in the literature with regards to the use of CB stem cells to neurological applications including in vitro work, pre-clinical animal studies, and patient clinical trials.     

Ex Vivo Generated Human Cord Blood Myeloid-Derived Suppressor Cells Attenuate Murine Chronic Graft-versus-Host Diseases

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with anti-inflammatory activity, and expanded murine MDSCs are capable of attenuating preclinical acute graft-versus-host disease (aGVHD) severity. Two murine cGVHD models were used to evaluate the effectiveness of ex vivo cultured human cord blood (hCB) MDSCs in chronic GVHD (cGVHD). First, GVHD recipients surviving in a classic C57BL/6 into MHC-mismatched BALB/c aGVHD model developed cGVHD. Second, donor pretreatment with granulocyte colony-stimulating factor (G-CSF) induced cGVHD. hCB-MDSCs (1?×?10⁶) were intravenously injected to determine their preventive effects (on days 5, 7, 10, and 21) or therapeutic effects (on days 21, 28, and 35). In the first model the onset of clinical cutaneous cGVHD was significantly delayed in preventive hCB-MDSCs–treated allogeneic recipients. Pathologic scoring of target organs confirmed these clinical results. Importantly, thymic tissues of GVHD mice treated with hCB-MDSCs were less severely damaged, showing higher numbers of double (CD4 and CD8) positive T cells with reduced expansion of donor-type CD4 and CD8 T cells. Moreover, late infusion of hCB-MDSCs controlled the severity of established cGVHD that had occurred in control recipients. In the second model, cGVHD induced by G-CSF–mobilized stem cell graft was associated with promotion of Th 17 and Th 2 differentiation. hCB-MDSCs attenuated clinical and pathologic cGVHD severity. Increased production of IL-17 and more infiltration of T cells and macrophages in cGVHD mice were markedly reduced after hCB-MDSCs treatment. Importantly, Foxp3⁺ regulatory T cells and IFN-γ–producing T cells were expanded, whereas IL-17– and IL-4–producing T cells were decreased in allogeneic recipients of hCB-MDSCs. Taken together, these results showed that hCB-MDSCs have preclinical capability of attenuating cGVHD by preserving thymus function and regulating Th 17 signaling, suggesting a possible therapeutic strategy for clinical application.     

Role of Interleukin-15 in Umbilical Cord Blood Transplantation

Owing to its easier accessibility and less severe graft-versus-host disease, umbilical cord blood (UCB) has been increasingly used as an alternative to bone marrow for hematopoietic stem-cell transplantation. Naiveté of UCB lymphocytes, however, results in delayed immune reconstitution and infection-related mortality in transplant recipients. This article reviews UCB immunology and addresses the potential therapeutic role of interleukin (IL)-15, a pleiotropic γ chain signaling cytokine, in modulating immune reconstitution, graft-versus-host disease (GVHD), graft-versus-leukemia effect, and infection susceptibility during the post-UCB transplant period. Cytokine immunotherapy using IL-15 simultaneously modulates several immune compartments, thus holds promise for facilitating post-transplant recovery and augmenting antitumor effect without aggravating GVHD in the setting of UCB transplantation.     

Comparative Analysis of the Immunophenotypes of Decidual and Peripheral Blood Large Granular Lymphocytes and T Cells During Early Human Pregnancy

PROBLEM : The functional role of the leukocytes in the decidua is not clear. They may regulate the maternal immune response to the fetal allograft. However, the factors controlling maternal and fetal communication have not yet been identified. METHOD : A comparative analysis of the phenotypes of decidual and peripheral blood large granular lymphocytes (LGLs) and T lymphocytes in early human pregnancy was performed on decidual tissue and blood samples obtained from ten patients at therapeutic abortion. RESULTS : Whereas most of the decidual LGLs were found to have a CD56^bright++ phenotype, most of the peripheral blood NK cells (90%) showed the classical CD56^dim+ phenotype, and only a small proportion were CD56^bright+ cells. Another striking difference was found in the expression of very late antigen 1 (VLA-1, CD49a): Almost all the decidual CD56^bright++ LGLs, but virtually none of the peripheral blood CD56⁺ NK cells expressed this antigen. Further differences were found in the expression of CD16, CD44, CD45RA, CD54, and CD57. There were also differences in phenotype between T cells derived from decidual tissue and those derived from peripheral blood. Approximately 31% of the CD3⁺ decidual T cells expressed VLA-1, but this antigen was virtually absent on peripheral blood T cells. A further difference was seen in the expression of HLA-DR. This activation antigen was found on 32 ± 13% of the decidual T cells but only 8 ± 5% of the peripheral blood T cells. Additionally, the proportion of cells expressing CD38 was higher among decidual than peripheral blood T cells. CONCLUSION : The findings suggest that both decidual LGLs and a subset of decidual T cells are activated and possibly play a role in the control of trophoblast growth and placental development.     

Th17 and Th1 Lymphocytes Are Correlated with Chronic Periodontitis

T cells are involved in the homeostasis of periodontal tissues and mediate bone loss in periodontitis, but the involvement of T-helper cells in chronic periodontitis (CP) in a Chinese population is still unclear. This study aimed to assess the distribution of peripheral and local T helper (Th17) and Th1 in CP. Sixty-eight patients with CP and 43 healthy controls were recruited from April 2012 to July 2014 at the Department of Stomatology, People’s Hospital of Xinjiang Uygur Autonomous Region (China). The proportions of Th17 (CD3⁺CD4⁺IL-17⁺) and Th1 (CD3⁺CD4⁺IFN-γ⁺) T-cells in peripheral blood samples were assessed by flow cytometry. Immunohistochemistry was used to quantify interleukin-17 (IL-17) and interferon-gamma (IFN-γ) protein levels in gingival biopsy samples. mRNA levels of IL-17, IFN-γ RORγt, and T-bet in gingival biopsy samples were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The proportions of circulating Th17 cells and Th1 cells were both more abundant in CP patients than in controls (Th17: 1.05% ± 0.87% vs. 0.62% ± 0.49%, P < 0.01; Th1: 13.93% ± 7.94% vs. 8.22% ± 4.50%, P < 0.001). Positive correlations were obtained between the proportion of circulating Th17 cells and probing depth (PD) (r = 0.320, P = 0.001) and between the proportion of circulating Th1 cells and PD (r = 0.372, P < 0.001). IL-17 and IFN-γ protein levels in gingival biopsy samples were markedly increased in CP compared to controls (both P < 0.05). Relative IFN-γ, IL-17A, and T-bet mRNA levels in CP biopsies were higher compared to controls (all P < 0.05). These results suggest that elevated peripheral and local Th17 and Th1 cells might be involved in the pathogenesis of CP.     

脐血和不同年龄组成人红细胞聚集、变形能力的分析

目前国内对心、脑血管等疾病的红细胞流变学研究较多,而对新生儿脐血的红细胞聚集、变形尚未见报道。我们测定了２０例足月新生儿脐带血和１１５例不同年龄健康成人的红细胞聚集、变形指数,并进行比较分析,以探讨新生儿红细胞聚集、变形能力的特点及增龄对成人红细胞流变性的影响。１　资料和方法１．１　对象脐血组：足月健康新生儿２０例,男性９例、女性１１例;成人组：健康成人１１５例,男性５４例、女性６１例,按不同年龄分组,１组１８～２９岁,２组３０～３９岁,３组４０～４９岁,４组５０～５９岁,５组＞６０岁。经体检均…     

Human Fetal/Neonatal Suppressor Activity: Relation Between OKT Phenotypes and Sensitivity to Prostaglandin E2 in Maternal and Neonatal Lymphocytes

ABSTRACT: T lymphocytes from human fetuses and newborns strongly and spontaneously suppress various adult cell functions (i.e. T-cell proliferation, B-cell differentiation, and Ig synthesis). The precise phenotype of the suppressor cell is controversial. In this investigation we use cord T-cell subsets negatively selected by the panning technique or by complement-mediated lysis using the monoclonal antibodies OKT4 and OKT8. Cord T cells deprived of the OKT4⁺ subpopulation exerted only a marginal suppressor activity (12 ± 7 as compared to 73 ± 4% of unfractionated T cells) on the proliferation of maternal cells in our PHA-stimulated co-culture assay using sex chromosomes as markers for dividing cord (male) and maternal cells. The suppressive effect was direct, i.e. not mediated by induction of maternal OKT8⁺ suppressor effector cells. Cord and maternal T-cell subsets were also tested for their sensitivity to exogenous prostaglandin E₂ (PGE₂) at doses varying between 1.4 × 10^?5 and 1.4 × 10^?9 M. Both maternal OKT4^? and OKT8^? T-cell subsets were highly sensitive to suppression by PGE₂. In contrast, cord OKT8^? T cells were essentially nonsensitive at all doses of PGE₂ used, whereas cord OKT4^? T cells were significantly suppressed at four out of five concentrations tested (1.4 × 10^?6 through 1.4 × 10^?9). Our results suggest a direct correlation between the phenotypes of the cord-suppressor and maternal-target T cells and their sensitivity to PGE₂.     

人脐血内皮祖细胞的分离、培养及鉴定

目的探索人脐静脉血内皮祖细胞的分离培养,为内皮祖细胞的临床应用提供实验方法。方法选择脐静脉血,应用密度梯度离心法,获取单个核细胞,接种于预先包埋了人纤维连接蛋白的培养板,用加入生长因子VEGF165和bFGF的内皮细胞专用培养基EGM-2MV培养细胞,3d后,洗掉非贴壁细胞,换培养液继续培养至7d,收集贴壁细胞进行细胞分析。激光共聚焦显微镜进行细胞功能学鉴定,流式细胞术测定祖细胞和内皮细胞系标志。MTT比色法检测细胞的生长状态。结果经过梯度密度离心和贴壁法选择的细胞能特异性吸附FITC标记的荆豆凝集素并内吞DiI-acLDL,祖细胞标志CD133及内皮细胞特异性抗原CD34、KDR检测,其阳性率分别为（27．05±2．94）％、（16．37±2．69）％和（56．67±7．29）％;体外培养的内皮祖细胞具有良好的细胞增殖活性。结论人脐静脉血中可以分离培养内皮祖细胞,为内皮祖细胞的进一步研究及临床应用奠定了基础。     

Effect of ABO Blood Group Incompatibility on the Outcome of Single-Unit Cord Blood Transplantation after Myeloablative Conditioning

ABO blood group incompatibility between donor and recipient has been associated with poor transplant outcomes in allogeneic hematopoietic stem cell transplantation. However, its effect on the outcome of cord blood transplantation (CBT) has yet to be clarified. We retrospectively analyzed 191 adult patients who received single-unit CBT after myeloablative conditioning for malignant disease in our institute. Major mismatch showed a significantly lower incidence of platelet engraftment compared with ABO match as a reference (hazard ratio, .57; P = .01). Nevertheless, there was no increase in graft-versus-host disease, transplant-related mortality, and overall mortality after ABO-incompatible CBT. These data suggested that donor–recipient ABO incompatibility does not have a significant impact on outcome after myeloablative CBT for hematological malignancies.