Dynamics of CD4^＋CD25^＋ T Cells in Spleens and Mesenteric Lymph Nodes of Mice Infected with Schistosomajaponicum

CD4~ CD25~ T cells play a major role in modulating immune response,but few reports havebeen published about schistosomiasis.Here,we investigated the changes in CD4~ CD25~ T cell populations inspleens and mesenteric lymph nodes of mice infected with Schistosoma japonicum.The proportions ofCD4~ CD25~ T cells in total CD4~ T cells were analyzed by flow cytometry.CD25 and Foxp3 expression wasmeasured by real-time quantitative polymerase chain reaction.The suppressive activities of CD4~ CD25~ Tcells were detected by in vitro proliferation of splenocytes.Evidence showed that the percentage of CD4~ CD25~ T cells was the same as controls 3 weeks post-infection.At the acute stage of infection,the percentagedecreased significantly.However,at the chronic stage of infection,it rebounded to normal levels or evenhigher.The expression of the CD25 and Foxp3 showed gradual increase along with the infection progress.Invitro experiment also showed the strong suppressive effect of CD4~ CD25~ T cells,isolated during the chronicstage,on proliferation of the CD25~-splenocytes.This is the first time that the dynamics of CD4~ CD25~ T cellpopulations was demonstrated in mice infected with schistosomiasis.In conclusion,our data indicated thatCD4~ CD25~ cells might be involved in the immune modulation during S.japonicum infection,which en-hances current knowledge of the mechanisms of the immuno-downregulation and re-infection inschistosomiasis.     

Downregulation of CD4＋CD25＋ regulatory T cells may underlie enhanced Th1 immunity caused by immunization with activated autologous T cells

Regulatory T cells （Treg） play important roles in immune system homeostasis, and may also be involved in tumor immunotolerance by suppressing Th1 immune response which is involved in anti-tumor immunity. We have previously reported that immunization with attenuated activated autologous T cells leads to enhanced anti-tumor immunity and upregulated Thl responses in vivo. However, the underlying molecular mechanisms are not well understood. Here we show that Treg function was significantly downregulated in mice that received immunization of attenuated activated autologous T cells. We found that Foxp3 expression decreased in CD4＋CD25＋ T cells from the immunized mice. Moreover, CD4＋CD25＋Foxp3＋ Treg obtained from immunized mice exhibited diminished immunosuppression ability compared to those from naive mice. Further analysis showed that the serum of immunized mice contains a high level ofanti-CD25 antibody （about 30 ng/ml, p〈0.01 vs controls）. Consistent with a role ofanti-CD25 response in the downregulation of Treg, adoptive transfer of serum from immunized mice to naive mice led to a significant decrease in Treg population and function in recipient mice. The triggering of anti-CD25 response in immunized mice can be explained by the fact that CD25 was induced to a high level in the ConA activated autologous T cells used for immunization. Our results demonstrate for the first time that immunization with attenuated activated autologous T cells evokes anti-CD25 antibody production, which leads to impeded CD4＋CD25＋Foxp3＋ Treg expansion and function in vivo. We suggest that dampened Treg function likely contributes to enhanced Thl response in immunized mice and is at least part of the mechanism underlying the boosted anti-tumor immunity.     

A glucocorticoid amplifies IL-2-induced selective expansion of CD4^＋CD25^＋FOXP3^＋ regulatory T cells in vivo and suppresses graft-versus-host disease after allogeneic lymphocyte transplantation

Regulatory T （Treg） cells are a subpopulation of T cells that not only prevent autoimmunity, but also control a wide range of T cell-dependent immune responses. Glucocorticoid treatment （dexamethasone, or Dex） has been reported to amplify IL-2-mediated selective in vivo expansion of Treg cells. We simultaneously administered Dex and IL-2 to the donor in a murine allogeneic lymphocyte transplantation model to expand functional suppressive CD4＋CD25＋FOXP3＋ T cells in the graft and to raise the regulatory T cell/effector T cell （Treg/ Teff） ratio to prevent graft-versus-host disease （GVHD）. After combined treatment of the donor with Dex （5 mg/kg/day） and IL-2 （300,000 IU/mouse/day） for 3 days, grafts were subjected to flow cytometric analysis, and transplantation was carried out from male C57BL/6 mice to female BALB/c mice aged 8-12 weeks. Results showed that short-term simultaneous administration of Dex and IL-2 markedly expanded functional suppressive CD4＋CD25＋FOXP3＋ T cells in the murine spleen. In this murine allogeneic transplantation model, the grafts from donors with Dex and IL-2 pre-treatment led to a longer survival time for the recipients than for the control group （median survival time 〉 60 day vs. 12 day, P = 0.0002）. The ratio of Treg/Teff also increased remarkably （0.43 ±0.15 vs. 0.14 ± 0.01, P = 0.01）. This study demonstrated that co-stimulation with Dex and IL-2 selectively expanded functional CD4＋CD25＋FOXP3＋ T cells in vivo, and that grafts from donors pre-treated with Dex and IL-2 led to longer survival time and greater suppression of GVHD after allogeneic transplantation. Thus, GVHD can be suppressed by the specific expansion of regulatory T cells with Dex and IL-2 in graft donors.     

Selective support of a mouse thymus epithelial cell line （MTEC1） to the viability and proliferation of CD4＋CD8—,CD4^—CD8^—,and CD4^＋CD8^＋thymocytes in vitro

The MTEC1 cell line,established in our laboratory,is a normal epithelial cell line derived from thymus medulla of Balb/c mice and these cells constituteively produce multiple cytokines.The selection of thymic microenvironment on developing T cells was investigated in an in vitro system.Unseparated fresh thymocytes from Balb/c mice were cocultured with MTEC1 cells or/and MTEC1-SN,then,the viability,proliferation and phenotypes of cultured thymocytes were assessed.Without any exogenous stimulus,both MTEC1 cells and MTEC1-SN were able to maintain the viability of thymocytes,while only the MTEC1 cells,not the MTEC1-SN,could directly activate thymocytes to exhibit moderate proliferation,indicating that the proliferative signal is delivered through cell surface interatcions of MTEC1 cells and thymocytes.Phenotype analysis on FACS of viable thymocytes after coculture revealed that MTEC1 cells preferentially activate the subsets of CD4^ CD8^-,CD4^ CD^8 and CD^4- CD^8- thymocytes;whereas MTEC1-SN preferentially maintained the viability of CD4^ CD^8- and CD4^-CD8^ thymocyte subsets.For the Con A-activated thymocytes.both MTEC1 cells and MTEC1-SN provided accessory signal(s) to significantly increase the number of viable cells and to markedly enhance the proliferation of thymocytes with virtually equal potency,phenotyped as CD4^ CD8^-,CD4^-CD8^ ,and CD^4-CD8^-subests,In summary,MTEC1 cells displayed Selection of thymic epithelial cells on thymocyte subsets. selective support to the different thymocyte subsets,and the selectivity is dependent on the status of thymocytes.     

TGF—β receptors in mouse ES—5 cells and their differentiated derivatives

By radioreceptor binding studies with iodinated TGF－β1,it has been shown that an undifferentiated ES-5 cell expresses approximately 3270 receptors with a dissociation constant Kd-130pM,but after the induction of differentiation by retinoic acid and dBcAMP,the receptor number of a differentiated RA-ES-5 cell was increased about 80% and the Kd was also increased to 370 pM.Furthermore,more direct evidence supporting the expression of TGF-β type I and type Ⅱ receptors in both ES-5 and RA-ES-5 cells has come from dot blot hybridization of cellular mRNA with cDNA probes for type I and type Ⅱ receptors.Meanwhile,mRNA expression level of types I and Ⅱ receptors in R-ES-5 cells were higher than that in ES-5 cells.Down-regulation of TGF-β receptors with a significant decrease in the rate of cell proliferation in both cells,was found by employing a pretreatment with neutralizing antibody to TGF-β1.The possible role of receptors for TGF-β in cell differentiation is discussed here.     

Response of T cells in vivo induced by repeated superantigen treatments at different time intervals

We have investigated the response of T cells to staphylococcal enterotoxin A （SEA） injections in vivo. We found that a single injection of SEA with an optimal dose of 10μg increased the expression of both CD4 and CD8 significantly. There was expansion of SEA-reactive T cells in vivo after SEA re-injection and the time interval between injections strongly influenced the responsiveness of CD4^＋ and CD8^＋ T cells. Anergy of T cells was observed after three SEA treatments. The time interval between injections mainly affected the unresponsiveness of CD4^＋ T cells, not CD8^＋ T cells. Marked deletion followed by anergy of CD4^＋ T cells was induced at short intervals, and anergy without obvious deletion of CD4^＋ T cells was induced at long intervals. We also found that the anergic state was reversible in vivo. Repeated SEA stimulation led to down-regulation of interleukin （IL）-2, and high levels of IL-10. This study showed that both CD4^＋ and CD8^＋ SEA-primed T cells were responsive to SEA rechallenge in vivo, and a third injection was needed to induce the anergy of T cells.     

Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference

Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation.Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or after malignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper we demonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression in airway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targeting human IL-8 in cultured airway epithelial cells (IB3-1, Cftr; C38, Cftr-corrected) stimulated with TNF-α, IL-1β or heat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reduced by shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels of IκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for the treatment of inflammatory diseases.     

Glycogen synthase kinase-3β positively regulates the proliferation of human ovarian cancer cells

Although glycogen synthase kinase-3 （GSK-3） might act as a tumor suppressor since its inhibition is expected to mimic the activation of Wnt-signaling pathway, GSK-3β may contribute to NF-κB activation in cancer cells leading to increased cancer cell proliferation and survival. Here we report that GSK-3β activity was involved in the proliferation of human ovarian cancer cell both in vitro and in vivo. Inhibition of GSK-3 activity by pharmacological inhibitors suppressed proliferation of the ovarian cancer cells. Overexpressing constitutively active form of GSK-3β induced entry into the S phase, increased cyclin D1 expression and facilitated the proliferation of ovarian cancer cells. Furthermore, GSK-3 inhibition prevented the formation of the tumor in nude mice generated by the inoculation of human ovarian cancer cells. Our findings thus suggest that GSK-3β activity is important for the proliferation of ovarian cancer cells, implicating this kinase as a potential therapeutic target in ovarian cancer.     

Neural progenitor cells from human induced pluripotent stem cells generated less autogenous immune response

The breakthrough development of induced pluripotent stem cells(iPSCs)raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells.However,whether iPSC-derived functional cell lineages generate a deleterious immune response upon auto-transplantation remains unclear.In this study,we differentiated five human iPSC lines from skin fibroblasts and urine cells into neural progenitor cells(NPCs)and analyzed their immunogenicity.Through co-culture with autogenous peripheral blood mononuclear cells(PBMCs),we showed that both somatic cells and iPSC-derived NPCs do not stimulate significant autogenous PBMC proliferation.However,a significant immune reaction was detected when these cells were co-cultured with allogenous PBMCs.Furthermore,no significant expression of perforin or granzyme B was detected following stimulation of autogenous immune effector cells(CD3+CD8 T cells,CD3+CD8+T cells or CD3 CD56+NK cells)by NPCs in both PBMC and T cell co-culture systems.These results suggest that human iPSC-derived NPCs may not initiate an immune response in autogenous transplants,and thus set a base for further preclinical evaluation of human iPSCs.     

TGF-β1-promoted epithelial-to-mesenchymal transfor mation and cell adhesion contribute to TGF-β1-enhanced cell migration in SMMC-7721 cells

Transforming growth factor-bl (TGF-β1), a multi-function polypeptide, is a double-edged sword in cancer. For some tumor cells, TGF-β1 is a potent growth inhibitor and apoptosis inducer. More commonly, TGF-β1 losesits growth-inhibitory and apoptosis-inducing effects, but stimulates the metastatic capacity of tumor cells. It is currently little known about TGF-β1-promoted cell migration in hepatocellular carcinoma (HCC) cells, let alone its mechanism. In this study, we found that TGF-β1 lost its tumor-suppressive effects, but significantly stimulated cellmigration in SMMC-7721 human HCC cells. By FACS and Western blot analysis, we observed that TGF-β1 enhanced the expression of ct5131 integrin obviously, and subsequently stimulated cell adhesion onto fibronectin(Fn). Furthermore, we observed that TGF-β1 could also promote SMMC-7721 cells adhesion onto laminin (Ln).Our data also provided evidences that TGF-β1 induced epithelial-to-mesenchymal transformation (EMT) in SMMC-7721 cells. First, SMMC-7721 cells clearly switched to the spindle shape morphology after TGF-β1 treatment.Furthermore, TGF-β1 induced the down-regulation of E-cadherin and the nuclear translocation of β1-catenin. These results indicated that TGF-β1-promoted cell adhesion and TGF-β1-induced epithelial-to-mesenchymal transfor-mation might be both responsible for TGF-β1-enhanced cell migration.     

Different roles of TGF-β in the multi-lineage differentiation of stem cells

Stem cells are a population of cells that has infinite or long-term self-renewal ability and can produce various kinds of descendent cells.Transforming growth factor β(TGF-β) family is a superfamily of growth factors,including TGF-β1,TGF-β2 and TGF-β3,bone morphogenetic proteins,activin/inhibin,and some other cytokines such as nodal,which plays very important roles in regulating a wide variety of biological processes,such as cell growth,differentiation,cell death.TGF-β,a pleiotropic cytokine,has been proved to be differentially involved in the regulation of multi-lineage differentiation of stem cells,through the Smad pathway,non-Smad pathways including mitogen-activated protein kinase pathways,phosphatidylinositol-3-kinase/AKT pathways and Rholike GTPase signaling pathways,and their cross-talks.For instance,it is generally known that TGF-β promotes the differentiation of stem cells into smooth muscle cells,immature cardiomyocytes,chondrocytes,neurocytes,hepatic stellate cells,Th17 cells,and dendritic cells.However,TGF-β inhibits the differentiation of stem cells into myotubes,adipocytes,endothelial cells,and natural killer cells.Additionally,TGF-β can provide competence for early stages of osteoblastic differentiation,but at late stages TGF-β acts as an inhibitor.The three mammalian isoforms(TGF-β1,2 and 3) have distinct but overlapping effects on hematopoiesis.Understanding the mechanisms underlying the regulatory effect of TGF-β in the stem cell multi-lineage differentiation is of importance in stem cell biology,and will facilitate both basic research and clinical applications of stem cells.In this article,we discuss the current status and progress in our understanding of different mechanisms by which TGF-β controls multi-lineage differentiation of stem cells.     

Effect of Human WEE1 and Stem Cell Factor on Human CD34^＋ Umbilical Cord Blood Cell Damage Induced by Chemotherapeutic Agents

Myelosuppression is one of the major side-effects of most anticancer drugs. To achieve myeloprotection, one bicistronic vector encoding anti-apoptotic protein human WEE l （WEElHu） and proliferation-stimulating stem cell factor （SCF） was generated. In this study, we selected human umbilical cord blood CD34^＋ cells as the in vitro model in an attempt to investigate whether WEEIHu, rather than conventional drug-resistant genes, can be introduced to rescue cells from the damage by chemotherapeutic agents such as cisplatin, adriamycin, mitomycin-c and 5-fluorouracil. Cell viability and cytotoxicity assay, colony-forming units in culture assay and externalization of phospholipid phosphatidylserine analysis showed that the expression of WEElHu and SCF in CD34^＋ cells provided the cells with some protection. These findings suggest that the expression of WEElHu and SCF might rescue CD34^＋ cells from chemotherapyinduced myelosuppression.     

A NEW CELL CLONE DERIVED FROM TRICHOPLUSIA NI TN5B1-4 CELLS

The characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. There are numerous selective and adaptive culture processes, in addition to genetic instability, that may improve phenotypic change in cell growth, virus susceptibility, gene expression, and production of virus. Similar detrimental effects of long term passaging of insect cells have also been reported for continuous cell lines, for example, Tn5B 1-4 cells, which are the most widely used for the baculovirus expression vector system (BEVS), provide superior production of recombinant proteins,however, this high productivity may be more evident in low passage cells. In this paper, we describe the isolation of a cell clone, Tn5B-40, from low passage Tn5B 1-4 cells. The growth characteristics,productions of virus, and high level of recombinant protein productions were determined. The results showed the susceptibility of both clone and Tn5B 1-4 cells to wild-type AcNPV was approximately the same rate with over 95% of infection; when the cloned cells were infected with recombinant baculoviruses expressing β-galactosidase and secreted alkaline phosphatase (SEAP), expression of the recombinant proteins from the cloned cells exceeded that from the parental Tn5B 1-4 cells.     

Expression of Epitope-Tagged SYN3 Cohesin Proteins Can Disrupt Meiosis in Arabidopsis

a-kleisins are core components of meiotic and mitotic cohesin complexes. Arabidopsis contains genes encoding four a-kleisins. SYN1, a REC8 ortholog, is essential for meiosis, while SYN2 and SYN4 appear to be SCCI orthologs and function in mitosis. SYN3 is enriched in the nucleolus of meiotic and mitotic cells and is essential for megagametogenesis. It was recently shown that expression of SYN3-RNAi constructs in buds cause changes in meiotic gene expression that result in meiotic alterations. In this report we show that expression of SYN3 from the 35S promoter with either a c-terminal Myc or FAST tag causes a reduction in SYN1 mRNA levels that results in al- terations in sister chromatid cohesion, homologous chromosome synapsis female meiosis. and synaptonemal complex formation during both male and