Mesenchymal stem cells inhibit the expression of CD25 (interleukin-2 receptor) and CD38 on phytohaemagglutinin-activated lymphocytes

Mesenchymal stem cells (MSC) are immunomodulatory and inhibit lymphocyte proliferation. We studied surface expression of lymphocyte activation markers and secreted cytokines, when lymphocytes were activated in the presence of MSC. MSC suppressed the proliferation of phytohaemagglutinin (PHA)-stimulated CD3+, CD4+ and CD8+ lymphocytes. MSC significantly reduced the expression of activation markers CD25, CD38 and CD69 on PHA-stimulated lymphocytes. Mixed lymphocyte culture (MLC) supernatants containing MSC suppressed proliferation of MLC and PHA-stimulated lymphocytes dose-dependently. MSC secrete osteoprotegerin (OPG), but not hepatocyte growth factor (HGF) or transforming growth factor-beta (TGF-beta). Stromal-cell-derived factor-1 (SDF-1) is not expressed on the cell surface. A recent report suggested that T-cell suppression by MSC is mediated by HGF and TGF-beta. MSC suppression was not restored by the addition of neutralizing antibodies against SDF-1, OPG, HGF or TGF-beta, alone or in combination. Addition of guanosine to PHA-stimulated lymphocyte cultures containing MSC did not affect lymphocyte proliferation. The immunosuppressive effects of cyclosporine and MSC did not interfere, when present in the cultures of PHA-activated lymphocytes. In summary, human MSC suppress proliferation of both CD4+ and CD8+ lymphocyte and decrease the expression of activation markers.     

骨髓间充质干细胞对致脑脊髓炎性MBP68-86特异性T淋巴细胞的调节作用

目的 探讨骨髓间充质干细胞(MSCs)对致脑脊髓炎性MBP68-86-特异性T淋巴细胞的抑制和激活作用.方法 全骨髓贴壁法提取、培养Lewis大鼠股骨、胫骨内的MSCs;建立实验性自身免疫性脑脊髓炎(EAE)动物模型,对照组大鼠仅免疫完全弗氏佐剂(CFA);EAE大鼠免疫10～11 d后,MBP68-86特异性T淋巴细胞被取出进行体外T淋巴细胞增值试验.从Lewis大鼠中得到的间质干细胞分别按下列要求预培养:在24孔板中与T细胞比值:1:1(1×106骨髓基质细胞/T细胞1×106)1:5、1:10、1:50或1:100;ELISA方法检测淋巴细胞培养上清液.结果 同种同基因的MSCs以MSCs与效应T细胞比例为1:1、1:5、1:10时共培养能够显著抑制MBP68-86特异性T淋巴细胞的增殖能力,与没有加入MSCs的单独MBP68-86特异性T淋巴细胞组相比差异具有统计学意义(P＜0.01).结论 MSCs在高密度(MSCs/T细胞数比≥1:10)的情况下,对MBP68-86-特异性T细胞表现为抑制作用;在较低的密度(≤1:50)时,表现为刺激的作用.     

Immunosuppressive Properties of Mesenchymal Stem Cells Derived from Bone Marrow of Patients with Chronic Myeloid Leukemia

Mesenchymal stem cells (MSCs) have emerged as excellent candidates for clinical application because of their capabilities of differentiating into multiple mesenchymal lineages and supporting hematopoiesis. Recently, MSCs have gained further interests after the demonstration of an immunosuppressive role. However, it is still unclear whether the immunosuppressive capability of MSCs will be altered with disease state. In this study, we obtained and expanded MSCs from bone marrow of patients with chronic myeloid leukemia (CML). Our results showed that MSCs derived from CML do not express costimulatory molecules CD40, CD80, and CD86. When MSCs derived from CML were added back to T cells stimulated by mitogens, a significant inhibition of T-cell proliferation was evident. MSCs differentiated into various mesenchymal lineages did not alter their immunosuppressive effect on T-cell proliferation. A significant T-cell inhibition was found in a transwell system, in which cell-cell contact between MSCs and effector cells was prevented. Furthermore, we found that transforming growth factor β1 (TGF β1) and hepatocyte growth factor (HGF) were major mediators of T-cell suppression by MSCs derived from CML. These results demonstrated that autologous MSCs derived from CML could effectively suppress T-cell proliferation.     

Immunosuppressive properties of mesenchymal stem cells derived from bone marrow of patients with chronic myeloid leukemia

Mesenchymal stem cells (MSCs) have emerged as excellent candidates for clinical application because of their capabilities of differentiating into multiple mesenchymal lineages and supporting hematopoiesis. Recently, MSCs have gained further interests after the demonstration of an immunosuppressive role. However, it is still unclear whether the immunosuppressive capability of MSCs will be altered with disease state. In this study, we obtained and expanded MSCs from bone marrow of patients with chronic myeloid leukemia (CML). Our results showed that MSCs derived from CML do not express costimulatory molecules CD40, CD80, and CD86. When MSCs derived from CML were added back to T cells stimulated by mitogens, a significant inhibition of T-cell proliferation was evident. MSCs differentiated into various mesenchymal lineages did not alter their immunosuppressive effect on T-cell proliferation. A significant T-cell inhibition was found in a transwell system, in which cell-cell contact between MSCs and effector cells was prevented. Furthermore, we found that transforming growth factor beta1 (TGF beta1) and hepatocyte growth factor (HGF) were major mediators of T-cell suppression by MSCs derived from CML. These results demonstrated that autologous MSCs derived from CML could effectively suppress T-cell proliferation.     

间充质干细胞对混合淋巴细胞反应体系中T淋巴细胞的免疫调节作用

目的:探讨间充质干细胞(MSC)在体外对T淋巴细胞免疫调节作用的特点。方法:通过Ficoll梯度密度离心法分离出正常人骨髓单个核细胞,体外培养扩增MSC,获取第3代细胞。将其按照不同的比例加入双向混合淋巴细胞培养(MLC)体系中,在第3、5天,采用MTT比色法检测各组MLC中的T淋巴细胞的增殖情况,再用流式细胞术分析其与MSC共孵育前后细胞表面标记的变化情况。结果:加入了MSC的MLC体系中,MSC对T淋巴细胞的增殖抑制具有剂量依赖性,且随着时间延长,抑制程度增强;其中,CD4 T细胞亚群受抑不如CD8 T细胞亚群显著;另外,T淋巴细胞表面CD25的表达虽然较对照组有所下降,但是CD4、CD25共表达的细胞却较对照组有明显的上升趋势。T淋巴细胞表面活化抗原HLA-DR的表达较对照组有轻度减低。结论:MSC在体外能够明显抑制T淋巴细胞的增殖,其主要是针对CD8 T细胞(CTL)。另外,它还能下调活化T淋巴细胞上一些较特异的表面标记CD25和HLA-DR的表达。     

Canine and Equine Mesenchymal Stem Cells Grown in Serum Free Media Have Altered Immunophenotype

Mesenchymal stem cell (MSC) therapy is being increasingly used to treat dogs and horses with naturally-occurring diseases. However these animals also serve as critical large animal models for ongoing translation of cell therapy products to the human market. MSC manufacture for clinical use mandates improvement in cell culture systems to meet demands for higher MSC numbers and removal of xeno-proteins (i.e. fetal bovine serum, FBS). While serum-free media (SFM) is commercially available, its affects on MSC phenotype and immunomodulatory functions are not fully known. The objective of this study was to determine if specific MSC culture conditions, MSC expansion in HYPERFlasks® or MSC expansion in a commercially available SFM, would alter MSC proliferation, phenotype or immunomodulatory properties in vitro. MSCs cultured in HYPERFlasks® were similar in phenotype, proliferative capacity and immunomodulatory functions to MSCs grown in standard flasks however MSC yield was markedly increased. HYPERFlasks® therefore provide a viable option to generate greater cell numbers in a streamlined manner. Canine and equine MSCs expanded in SFM displayed similar proliferation, surface phenotype and inhibitory effect on lymphocyte proliferation in vitro. However, MSCs cultured in the absence of FBS secreted significantly less PGE₂, and were significantly less able to inhibit IFNγ secretion by activated T-cells. Immunomodulatory functions altered by expansion in SFM were species dependent. Unlike equine MSCs, in canine adipose-derived MSCs, the inhibition of lymphocyte proliferation was not principally modulated by PGE₂. The removal of FBS from both canine and equine MSC culture systems resulted in altered immunomodulatory properties in vitro and warrants further investigation prior to moving towards FBS-free culture conditions.     

CD4+CD25+Foxp3+IFN-γ+ human induced T regulatory cells are induced by interferon-γ and suppress alloresponses nonspecifically

Interferon-γ (IFN-γ)-producing CD3(+)CD4(+)CD25(+)Foxp3(+) peripheral blood lymphocytes (PBL) are more frequently detectable in patients with good than in patients with impaired long-term kidney graft function, suggesting an immunoregulatory role of this induced T regulatory (iTreg) subtype. Herein, the in vitro function of separated CD3(+)CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL that were induced by phorbol 12-myristate 13-acetate (PMA)/ionomycin or alloantigenic stimulation was investigated using cell coculture techniques and flow cytometry. CD4(+)CD25(+)Foxp3(+) PBL with intracellular IFN-γ production increased to 26% in cell cultures stimulated with PMA/ionomycin for 6 hours. Recombinant IFN-γ augmented and anti-IFN-γ monoclonal antibody blocked induction of CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL, suggesting their IFN-γ-dependent induction. In addition, CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL produced immunosuppressive interleukin (IL)-10, transforming growth factor-β, and IL-4 intracellularly and expressed both IFN-γ and IFN-γ receptors (CD119) on the cell surface, allowing separation of CD4(+)CD25(+)IFN-γ(+) PBL with 98% purity. Addition of enriched CD4(+)CD25(+)IFN-γ(+) PBL to autologous PMA/ionomycin stimulated PBL decreased blast formation (p < 0.05), indicating suppression of cell proliferation by CD4(+)CD25(+)IFN-γ(+) PBL. CD4(+)CD25(+)IFN-γ(+) PBL separated from primary mixed leukocyte cultures (MLC) and added to autologous or third-party secondary MLC suppressed allogeneic T-cell activation nonspecifically (p < 0.05). We conclude that CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL are induced by IFN-γ, making them sensors for IFN-γ and initial immune responses. Circulating CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL could suppress allogeneic T-cell responses in patients and may be involved in inhibition of the posttransplant alloresponse.     

Markers of cell death-activation in lymphocytes of vertically HIV-infected children naive to highly active antiretroviral therapy: the role of age.

BACKGROUND: Apoptosis plays a major role in depleting CD4(+) lymphocytes during infection with HIV-1. Few data exist on its role during HIV infection of children. Sensitivity of peripheral blood lymphocytes (PBLs) to apoptotic stimuli and the importance of the patient's age remain unclear. OBJECTIVES: We sought to analyze the following: (1) markers of cell death-activation (CD95, CD45 isoforms, and CD28) in PBLs from vertically HIV-infected children of different ages before highly active antiretroviral therapy; (2) changes in other PBL populations; (3) PBL sensitivity to cell death and mitochondrial damages; and (4) role of age during progression of infection. METHODS: Cell culture techniques and flow cytometry were used to analyze surface antigens, PBL susceptibility to apoptosis, or PBL susceptibility to change of mitochondrial membrane potential. RESULTS: Donor age had a strong negative correlation with numbers of CD4(+) and CD8(+) T cells. Virgin T lymphocyte (CD45RA(+), CD95(-)) levels and those of CD95(+) cells showed no correlation with the children's clinical status but did show a correlation with patient age. CD28(-) T lymphocytes were markedly augmented in HIV-infected children but were unrelated to stage of infection or age. A relevant decrease in B lymphocytes and an increase in natural killer cells were also found. Finally, PBLs from HIV-positive children had a marked tendency to undergo apoptosis and mitochondrial damage. CONCLUSION: Changes in PBL phenotype, increased expression of CD95, and high sensitivity to apoptosis suggest that a precocious aging of the immune system occurs in HIV-infected children.     

Signal transduction through crosslinking CD7 and IgM-Fc receptors that inhibits T-cell proliferation.

We have previously suggested a possible involvement of CD7 and/or the putative receptor for IgM-Fc (FcRmu) in the downregulation of T-cell responses to mitogen and alloantigen. In this report, we examined the effect of ligand-receptor interaction upon T-cell proliferation by using anti-CD7 monoclonal antibodies (mAbs) and purified human IgM (HIgM) and showed that crosslinking CD7 and/or FcRmu on T cells resulted in significant inhibition of mitogen- and alloantigen-induced proliferative responses. In most cases, crosslinking of FcRmu alone or together with CD7 receptors on peripheral blood lymphocytes (PBLs) resulted in more potent suppression of T-cell proliferation than that caused by crosslinking CD7 alone. Examination of potential inhibitory mechanisms revealed that crosslinking CD7 and FcRmu does not reduce cell viability or the expression of T-cell markers that are important for T-cell activation or proliferation. However, crosslinking CD7 and/or FcRmu of PBLs significantly reduced phytohemagglutinin (PHA)-induced IL-2 production and rendered PBLs unable to utilize exogenously added human recombinant IL-2 (rIL-2). Further examination of possible signal transduction that might be associated with crosslinking CD7 and/or FcRmu receptors indicated a marked reduction in the magnitude and duration of intracellular calcium (Ca++)i released in response to PHA. These findings suggest that crosslinking CD7 and FcRmu inhibits T-cell activation and proliferation by a calcium-dependent mechanism that inhibits IL-2 production and/or utilization.     

Regulation of p26-bcl-2 protein levels in human peripheral blood lymphocytes.

BACKGROUND: The protein encoded by the bcl-2 (B cell lymphoma/leukemia-2) proto-oncogene has been implicated in the regulation of lymphocyte cell survival and has been shown to interfere with programmed cell death (apoptosis). Recently, controversy has emerged regarding the expression of bcl-2 in circulating peripheral blood lymphocytes (PBL) and its correlation with lymphocyte proliferation. Using immunohistochemical methods, Pezzella et al. (Pezzella F, Tse G, Cordell J, Pulford K, Gatter K, Mason D. Am J Pathol 1990; 137:225-32) detected abundant amounts of Bcl-2 protein in resting PBLs and observed an inverse correlation between bcl-2 expression and cellular proliferation in lymphoid tissues. In contrast, previous in vitro studies of bcl-2 mRNAs showed that expression of this gene is very low in unstimulated PBLs but can be markedly induced when lymphocytes are stimulated to proliferate in culture (Reed JC, Tsujimoto Y, Alpers JD, Croce CM, Nowell PC. Science 1987; 236:1295-9; Granniger W, Seto M, Boutain B, Goldman P, Korsmeyer S. J Clin Invest 1987; 80:1512-5). EXPERIMENTAL DESIGN: To resolve this issue, the regulation of p26-Bcl-2 protein levels was examined in freshly isolated and in cultured human PBLs by immunoblotting using two different polyclonal antisera specific for the human Bcl-2 protein. RESULTS: When freshly isolated from whole blood, resting PBLs contained easily detectable amounts of p26-Bcl-2 protein that did not significantly change in culture when cellular proliferation was stimulated with mitogenic lectins and lymphokines. If processing of the blood was delayed, however, p26-Bcl-2 protein was low or undetectable in resting PBLs and underwent marked increases in its relative levels after in vitro stimulation of PBLs by mitogenic lectins and lymphokines. CONCLUSIONS: The findings indicate that bcl-2 is normally expressed in quiescent circulating lymphocytes, consistent with a role for this gene in the maintenance of lymphocyte survival, and illustrate the importance of correlating in vitro and in vivo expression data.     

失代偿期乙肝肝硬化患者的骨髓间充质干细胞的免疫调节作用

目的 观察失代偿期乙型肝炎肝硬化患者的骨髓间充质干细胞（BMSCs）对自体外周血淋巴细胞增殖及调节性T细胞（Tregs）亚群的影响.方法 从8例失代偿性肝硬化患者骨髓中分离培养MSCs,采用流式细胞仪分析鉴定MSCs的纯度,分离患者外周血淋巴细胞行羧基荧光素乙酰乙酸琥珀酰亚胺酯（CFDA SE）荧光染色,在植物血凝素（PHA）刺激下,培养基为自体血清,自体外周血淋巴细胞与BMSCs共培养,流式细胞术检测各组淋巴细胞增殖情况及CD4＋ CD25＋ CD127-Tregs频数.结果 流式细胞术显示：接触培养组及非接触培养组的细胞增殖较阳性对照组明显减低（P均＜0.01）,但亦显著高于阴性对照组（P均＜0.01）,其CD4＋ CD25＋ CD127-Tregs频率显著高于阳性及阴性对照组（P均＜0.01）;接触培养组与非接触培养组比较,淋巴细胞增殖及CD4＋ CD25＋ CD127-Tregs频数差异均无统计学意义（P均＞0.05）.结论 肝硬化患者BMSCs可抑制自体外周血淋巴细胞增殖、上调CD4＋ CD25＋ CD127-Tregs表达.     

Characterization and functionality of the CD200-CD200R system during mesenchymal stromal cell interactions with T-lymphocytes

Mesenchymal stromal cells (MSCs) possess a specific immunological profile that makes them potentially useful for immune-based therapies. Adipose tissue (AT) and Wharton's jelly (WJ) are considered to be valuable alternatives to bone marrow (BM) as sources of MSCs. These MSCs exhibit strong immunomodulatory properties that affect lymphocyte responses. The CD200/CD200R axis has been reported to be important in regulating the immune responses. Engagement of CD200R by CD200 initiates an inhibitory pathway that displays immunosuppressive effects. Because the CD200/CD200R axis is involved in immunoregulation, we investigated the expression and role of this ligand/receptor pair in MSCs and T-lymphocytes during co-culture. CD200 is differently expressed and modulated on MSCs depending on the tissue of origin and the culture conditions. Among the different MSC sources, WJ-MSCs express CD200 in the greatest proportion. This high constitutive CD200 expression may represent a distinctive marker for WJ-MSCs. A pro-inflammatory environment and IFN-γ in particular induce an increase in CD200 expression by BM-MSCs. In T-lymphocytes, CD200R and CD200 are differently distributed between the CD4(+) and CD8(+) T-cell subsets. During co-culture, blocking CD200-CD200R interactions does not prevent MSC-mediated inhibition of lymphocyte proliferation. However, depending on their origin, MSCs are able to modulate the expression of both CD200 and CD200R on some T-cells. Further study is required to understand the function of CD200 expression by nonmyeloid cells such MSCs and the significance of CD200 and C200R expression by T-cells. The findings presented here support bidirectional communication between MSCs and T-lymphocytes. Understanding the role of this ligand-receptor pair during co-culture will improve and increase the clinical use of MSCs.     

Suppression of natural killer cells in the presence of CD34+ blood progenitor cells and peripheral blood lymphocytes.

Abstract Mobilization of CD34 + peripheral blood progenitor cells (PBPCs) with granulocyte-colony stimulating factor (G-CSF) may induce functional alterations in peripheral blood lymphocyte (PBL) subsets. We and others have shown that natural killer (NK) cells from PBPC collections are less expandable in vitro than those obtained during steady-state hematopoiesis. We show here that the extent of this proliferation deficit is related to the number of circulating CD34 + cells in vivo at the time of PBPC apheresis. Likewise, addition of autologous CD34 + cells to unseparated PBL reduced the expansion of the NK-cell subset by 22.2% +/- 6.0% (n = 10; P <.005). In contrast, when using purified NK cells, their proliferation remained unimpaired by autologous CD34 + cells. Supernatants from CD34 + cells cultured with autologous PBLs had an inhibitory effect on proliferation of purified NK cells (n = 16; P =.03), indicating that an interaction between CD34 + cells and lymphocytes is essential for the suppressive effect on NK cells. To investigate the role of T cells in this interaction, intracellular cytokines were determined in T cells cultured for 7 days with or without autologous CD34 + cells. When cultured with CD34 + cells, the frequency of IL-2-producing CD4 + and CD8 + T cells was reduced by 19% and 24%, respectively, compared with T cells cultured alone (n = 7; P =.016). Interferon-gamma-producing T cells were slightly reduced ( P = not statistically significant [ns]). Finally, the influence of T cells and NK cells on the recovery of myeloid colony-forming cells (CFU-GMs) from purified CD34 + cells was examined. In the presence of T cells, 16% +/- 6% of the input CFU-GM recovered after 7 days, compared with 5% +/- 4% in the presence of NK cells (n = 5; P = ns). Our findings point to an inhibition of NK-cell proliferation mediated by an interaction of CD34 + cells and T cells occurring during PBPC mobilization with G-CSF.     

Mesenchymal stem cells expanded in human platelet lysate display a decreased inhibitory capacity on T- and NK-cell proliferation and function

The use of fetal bovine serum (FBS) for the culture and expansion of mesenchymal stromal cells (MSCs) limits their possible clinical applications. Although some recent studies recommended substituting FBS with human platelet lysate (HPL) for the expansion of MSCs for clinical use, the functional capacity of the expanded cells has only been partially explored. 10% FBS and two other commercial FBS-containing media (MesenCult and MesenPro) were compared with 10% HPL-containing medium for their ability to support MSCs expansion and immunomodulation. We demonstrate that HPL sustained MSC proliferation and expansion in vitro. However, the cumulative cell numbers recovered were comparable with those obtained in MesenPro medium. Moreover, we show that HPL alters the expression of some relevant MSC surface molecules, namely the DNAM-1 ligands PVR and Nectin-2, the NKG2D ligand ULBP3, the adhesion molecules CD49d and αvβ3 and the fibroblast-associated protein. In addition, MSCs cultured in HPL displayed impaired inhibitory capacity on T-cell proliferation to alloantigen and NK-cell proliferation and cytotoxicity. Finally, they showed decreased constitutive PGE2 production while IL-6, IL-8 and RANTES secretion were upregulated. These results imply some limitations in the use of HPL for the expansion of MSCs to be used as immunomodulators in clinical applications.     

Functional and phenotypic properties of peripheral T cells anergized by autologous CD3(+) depleted bone marrow cells

We have previously demonstrated that bone marrow cells (BMC) inhibit the generation of autologous Epstein-Barr virus (EBV) -specific cytotoxic T lymphocytes (CTL). It was also observed that CD3(+) cells obtained after 7 days of culture in the presence of autologous BMC could be used as inhibitors of EBV-CTL generation. In the present study, we examined these BMC induced regulator CD3(+) T cells with respect to phenotype, function, and T-cell activation pathways. We also questioned if the CD3(+) regulatory cell function is mediated by their direct effect on peripheral T cells or on the ability of antigen presenting cells (APC) to stimulate peripheral T cells. To answer this, CD3(+) cells from peripheral blood lymphocytes (PBL) were cultured with either CD3-depleted BMC or with CD3-depleted PBL. The CD3(+) cells were then isolated with immunomagnetic beads, designated as T(BM) and T(PBL), and were compared in functional studies. There was an increase in the expression of CD25 on T(BM) cells. The T(BM) cells also expressed less CD122 and a decreased number of CD3 molecules per cell. Both T(BM) and T(PBL) cell populations responded to mitogen (PHA) to the same magnitude. However, when stimulated through the CD3 complex with anti-CD3 monoclonal antibody (mAb), the T(BM) cells had a significantly decreased response than did T(PBL). The addition of IL-2 to these latter cultures augmented, but could not fully restore, the response. Additionally, stimulation of T(BM) cells with allogeneic cells failed to produce cytotoxic T cells. These "anergized" T(BM) and "nonanergized" (control) T(PBL) cells were added as third-party cells to a CTL generating culture of autologous PBL stimulated with allogeneic cells. The T(BM) cells exhibited suppressor function and inhibited the generation of CTL, in contrast with T(PBL). The effect of T(BM) cells on direct and indirect antigen presentation pathways demonstrated that T(BM) primarily effected indirect, but not direct, alloantigen presentation. To further explore the cytoplasmic T-cell activation events that occurred after the coculture of the PBL T cells with BMC, the levels of zeta-associated protein 70 (ZAP70) and extracellular receptor-activated kinase (ERK) were determined. There was a decrease in ZAP70 levels in the T(BM), which correlated with its reduced expression of cell surface CD3 and the attenuated response to anti-CD3 mAb activation. However, the activity of ERK was equally expressed by T(BM) and T(PBL). It, therefore, appears that the culturing of peripheral T cells with (non-T) BMC anergizes these cells (which become refractory to stimulation through the T-cell receptors), and induces immune suppressor function. These in vitro observations may provide a mechanism by which infused donor BMC serve to downregulate T-cell immunity.     

人脐血源性MSCs的免疫调节作用

目的 从人脐血中分离、培养间充质干细胞(MSCs)并探讨其对淋巴细胞的免疫调节作用.方法 淋巴细胞分离液分离人脐血单个核细胞,利用贴壁筛选法通过多次传代得到MSCs,流式细胞仪测定细胞表型;将获得的MSCs分别以不同数量加入到外周血混合淋巴细胞培养体系和植物血凝素(PHA)刺激的外周血淋巴细胞转化体系中,用H3-TdR标记β液体闪烁计数仪检测细胞增殖,观察脐血来源的MSCs对混合淋巴细胞反应和淋巴细胞转化的影响.结果 从人脐血中分离获得的贴壁细胞,呈成纤维样的细胞形态,CD29、CD105和CD166表达阳性,CD14、CD34和CD45表达阴性;人脐血源性MSCs在体外对混合淋巴细胞反应和PHA诱导的淋巴细胞转化均具有明显的抑制作用,抑制作用与细胞数量呈正相关.结论 从人脐血中可以成功地分离出MSCs,其对同种异体淋巴细胞具有免疫调节作用,为其作为骨组织工程异基因种子细胞来源打下基础.     

Multivariate analysis of differential lymphocyte cell cycle activity in Alzheimer's disease

Mounting evidence suggests cell cycle dysregulation is involved in the pathogenesis of Alzheimer's disease (AD) and that this failure is systemic, affecting not only neurons but also peripheral blood lymphocytes (PBLs). This study analyzed if differences in PBL proliferation activity could be used as a diagnostic biomarker for AD. CD69 and CD28 expressions on PBL T, B, and monocyte cells were measured by flow cytometry with and without mitogenic stimulation in healthy controls (HC), probable AD, and Parkinson's disease dementia (PDD) subjects. Univariate and multivariate scoring models were employed to evaluate the data relative to the clinical diagnoses. Eleven CD expression markers were significantly altered in AD subjects compared with a mixed pool of PDD and HC subjects using univariate models. Using multivariate models, seven CD expression markers were significantly altered in AD subjects compared with PDD subjects. Multivariate scoring demonstrated up to a 91% positive and 92% negative agreement with subject clinical diagnosis and had little correlation with the severity of dementia. Present findings suggest that with further development this analytical and multivariate modeling procedure could aid the current differential diagnosis of Alzheimer's disease.     

High mobility group box 1 protein inhibits the proliferation of human mesenchymal stem cells and promotes their migration and differentiation along osteoblastic pathway

Extracellular high mobility group box 1 (HMGB1) is a novel cytokine that takes part in the processes of inflammation, tissue damage and regeneration. Mesenchymal stem cells (MSCs) are adult stem cells characterized by their inherently suppressive activities on inflammative and allo-immune reactions. In the present study, we have addressed whether HMGB1 could affect the biological properties of human bone marrow MSCs. Transwell experiments showed that HMGB1 induced MSC migration and this effect could not be hampered by a blocking antibody against the receptor for advanced glycation end products (RAGE). MSCs exposed to HMGB1 were negative for CD31, CD45, CD80, and HLA-DR, and displayed equal levels of CD73, CD166, and HLA-ABC compared with their counterparts, but HMGB1 profoundly suppressed MSC proliferation in a dose-dependent manner as evaluated by carboxyfluorescein diacetate succinmidyl ester dye dilution assay. Furthermore, HMGB1 triggered the differentiation of MSCs into osteoblasts as identified by histochemical staining, traditional RT-PCR and real-time RT-PCR analysis on mRNA expression of lineage-specific molecular markers. The differentiation-inductive activity could neither be inhibited by RAGE neutralizing antibody. Moreover, HMGB1-treated MSCs displayed unchanged suppressive activity on in vitro lymphocyte cell proliferation elicited by ConA. Collectively, the data suggest that MSCs are a target of HMGB1.     

Clonal expansion of T cells following in vitro stimulation with autologous melanoma cells and interleukin-2 studied by molecular analysis of a T cell receptor.

Twelve autologous mixed peripheral blood (PBL) tumor cell interactions (MLTC) followed by in vitro expansion of the stimulated T cells in recombinant interleukin-2 (IL-2) were analyzed for the potential emergence of oligoclonal or monoclonal T cell populations in the PBL by Southern blot analysis of the T cell receptor (TCR) beta gene. The emergence of oligoclonal or monoclonal TCR beta gene rearranged populations was seen in 5 of the 12 cases. In 2 of these 5 cases only one dominantly rearranged band was observed. The emergence of oligoclonal or monoclonal T cell populations following stimulation with autologous melanoma cells was associated with predominant CD4 phenotype of the stimulated PBL exhibiting a varied degree of cytotoxicity toward the respective autologous melanoma cells. The evidence of emergence of monoclonal or oligoclonal T cell populations following stimulation with autologous tumor cells strongly supports the existence of T cell-mediated responses against autologous melanomas. Furthermore, cellular and molecular analyses of T cell responses in autologous mixed lymphocyte tumor cell interactions will provide valuable information on the nature of the T cell responses and on the pattern of gene segment usages by the T cells in response to the autologous tumor cells.     

Mesenchymal stem cells stimulate antibody secretion in human B cells

Mesenchymal stem cells (MSC) have immunomodulatory effects and inhibit T-cell responses to alloantigens and mitogens in vitro and in vivo. We wanted to examine the effect of MSC on human B cells. MSC stimulated IgG production, measured in an enzyme-linked immunospot (ELIspot) assay in blood and spleen lymphocytes. MSC only induced a low proliferation. When a semipermeable membrane separated MSC and mononuclear cells, the IgG production was stimulated in unfractionated lymphocytes. In contrast, enriched B cells required cell contact with MSC to produce IgG. Co-cultures of MSC and lymphocytes increased IFN-gamma production. MSC produce IL-6, and addition of MSC to spleen cells dramatically increased IL-6 levels. After lymphocyte stimulation with lipopolysaccharide (LPS), cytomegalovirus or varicella zoster virus, MSC either stimulated or inhibited IgG response, depending on the level of stimulation by LPS or the viral antigens. Similar results were obtained for enriched B cells. To conclude, MSC stimulate B-cell antibody secretion. The IgG secretion by activated B cells may be stimulated or inhibited by the addition of MSC, depending on the level of stimulation.