Abnormally high expression of BAFF on T lymphocytes from lung cancer-associated pleural effusions and its potent anti-tumor effect

In the present study, the expressions of B cell activating factor belonging to the tumor necrosis factor family （BAFF） and its receptors （BAFF-R and TACI） on T lymphocytes from malignant pleural effusion （MPE） were examined by fluorescence-activated cell sorting （FACS） analysis, and compared with those on the T lymphocytes from non-malignant pleural effusion （NMPE） and healthy controls. It was found that CD3 positive T lymphocytes （including CD4, CD8, and part of CD25 and CD69 positive cells） of MPE in lung cancer highly and consistently expressed the BAFF molecule, while high expressions of BAFF could only be found in phytohemagglutinin （PHA） or interleukin 2 （IL-2） induced T lymphocytes from NMPE or healthy controls. These results were consistent with the results from BAFF mRNA detection by real-time PCR. In addition, T lymphocytes from MPE expressed significantly more BAFF-R than those from NMPE or healthy controls, while the expression of TACI was increased on CD4＋ T cells but decreased on CD8＋ T cells when compared with controls. The Annexin/PI assay suggested that recombinant human BAFF （rhBAFF） could promote the survival rate of T lymphocytes from MPE, while the decoy receptor TACI-Fc fusion protein could promote the apoptosis rate of T lymphocytes. Cytokines in the supernatant detected by ELISA assay showed that rhBAFF could significantly upregulate the secretion of IFN-γ in vitro, and the IFN-γ level in the TACI-Fc-treated group resembled that of the control groups. All of these results indicated that the abnormally high expression of BAFF on T lymphocytes from MPE may play a role of antitumor effect.     

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.     

The immunologic and hematopoietic profiles of mesenchymal stem cells derived from different sections of human umbilical cord

Mesenchymal stem cells （MSCs） have been widely used in allogeneic stem cell transplantation. We compared im- munologic and hematopoietic characteristics of MSCs derived from whole human umbilical cord （UC）, as well as from different sections of UCs, including the amniotic membrane （AM）, Wharton＇s jelly （WJ）, and umbilical vessel （UV）. Cell phenotypes were examined by flow cytometry. Lymphocyte transformation test and mixed lymphocyte reaction were performed to evaluate the immuno-modulatory activity of MSCs derived from UCs. The mRNA expression of cytokines was detected by real- time polymerase chain reaction. Hematopoietic function was studied by co-culturing MSCs with CD34＋ cells iso- lated from cord blood. Our results showed that MSCs separated from these four different sections including UC, W J, UV, and AM had similar biological characteristics. All of the MSCs had multi-lineage differentiation ability and were able to differentiate into osteoblasts, adipocytes, and chondrocytes. The MSCs also inhibited the proliferation of allogeneic T cells in a dose-dependent manner. The relative mRNA expression of cytokines was examined, and the results showed that UCMSCs had higher interleukin-6 （IL6）, ILll, stem cell factor, and FLT3 expression than MSCs derived from specific sections of UCs. CD34＋ cells had high propagation efficiencies when co-cultured with MSCs derived from different sections of UCs, among which UCMSCs are the most efficient feeding layer. Our study demonstrated that MSCs could be isolated from whole UC or specific sections of UC with similar immuno- modulation and hematopoiesis supporting characteristics.     

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.     

Comparison of two in vitro angiogenesis assays for evaluating the effects of netrin-1 on tube formation

Netrin-1 is a neural guidance cue that also regulates vascu- lar development. Controversial results, however, have been obtained concerning the roles of netrin-1 in vascular devel- opment both in vivo and in vitro. In the present study, two in vitro angiogenesis assays were compared to evaluate the effects of netrin-1 secreted by retrovirally transduced mel- anoma cells （Mel2a-netrinl） on tube formation. The results showed that there was no obvious difference in tube forma- tion induced by conditioned media （CM） from the control, Mel2a-netrinl and Mel2a cells in a matrigel assay. The results of another in vitro assay, in which endothelial cells were co-cultured with human fibroblasts, however, showed that Mel2a-netrinl CM inhibited the tube formation, sup- posedly through blocking the elongation and coalescence of human umbilical vein endothelial cells （HUVECs）. These results confirmed that the matrigel assay is not able to demonstrate the anti-angiogenic roles of netrin-1.     

Human immune suppression is inducible by trichosanthin via CD8 cell—mediated pathway

Trichosanthin(Tk),a polypeptide with 249 amino acid residues isolated and purified from a Chinese medicinal herb,showed the capability of inducing abortion and was able to inhibit tumor growth and HIV replication.Owing to sequence homology of the peptide with a ribosomeinactivating protein,the downward activity of Tk was suggested to be related to its cytotoxic property.We report here,however,that Tk could exert potent inhibitory effects on human lymphoproliferative responses in vitro to allogeneic,mitogenic and soluble antigens with 50% inhibition doses ranged between 0.05 and 0.5μg／ml.The lowresponsivenesss caused by Tk was not due to toxic cytolysis.Rather,evidences suggested that,in the dose range adopted,the Tk-induced inhibition was attributable,at least in part,to immune suppression,in view of (1) Tk was more effective in the early stage of alloreactivity;(2)Suppression also occurred if responder cells were pulsetreated with Tk rather than cocultured;(3)Irradiated Tk-pulsed cells were capable of inducing suppression in a Tk-free culture;(4)Suppression could also be transferred by the supernatants of Tk-pulsed cultured cells;(5)Tk-induced immune suppression was diminished by depletion of CD8^ cells from the culture,and,finally;(6)Adding CD8^ cells back to the culture could restore the suppres sion.Thus the possibility that Tk might function as a down-regulator by immunological mechanisms in human immune responses is discussed.     

IL-9 enhances the growth of human mast cell progenitors under stimulation with stem cell factor

We examined the effects of IL-9 on human mast cell development from CD34(+) cord blood (CB) and peripheral blood cells in serum-deprived cultures. IL-9 apparently enhanced cell production under stimulation with stem cell factor (SCF) from CD34(+) CB cells. A great majority of the cultured cells grown with SCF + IL-9 became positive for tryptase at 4 wk. In methylcellulose cultures of CD34(+) CB cells, IL-9 increased both the number and size of mast cell colonies grown with SCF. Furthermore, SCF + IL-9 caused an exclusive expansion of mast cell colony-forming cells in a 2-wk liquid culture of CD34(+) CB cells, at a level markedly greater than for SCF alone. Clonal cell cultures and RT-PCR analysis showed that the targets of SCF + IL-9 were the CD34(+)CD38(+) CB cells rather than the CD34(+)CD38(-) CB cells. IL-9 neither augmented the SCF-dependent generation of progeny nor supported the survival of 6-wk-cultured mast cells. Moreover, there was no difference in the appearance of tryptase(+) cells and histamine content in the cultured cells between SCF and SCF + IL-9. The addition of IL-9 increased numbers of mast cell colonies grown with SCF from CD34(+) peripheral blood cells in children with or without asthma. It is of interest that mast cell progenitors of asthmatic patients responded to SCF + IL-9 to a greater extent than those of normal controls. Taken together, IL-9 appears to act as a potent enhancer for the SCF-dependent growth of mast cell progenitors in humans, particularly asthmatic patients.     

Expansion of CD34+ cells from human umbilical cord blood by FL and/or TPO gene transfected human marrow stromal cell lines

To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34+ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34+ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34+ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3±52.1)-fold, total progenitor cells (CFC) by (74.5±5.2)-fold and CD34+ cells by 15.7-fold.Maximal expansions of CFC and CD34+ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 ± 5.5)-fold and (57.0 ± 19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34+ cells within 28 days was found only in two combinations, I.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant difference between these two groups statistically. These results suggest that human umbilical cord blood CD34+ cells can be extensively expanded ex vivo by using gene transfected stromal cells along with cytokines.     

Regulation of the human WEE1Hu CDK tyrosine 15-kinase during the cell cycle.

In higher eukaryotes, the cyclin-dependent kinases (CDKs) are negatively regulated by phosphorylation on threonine 14 (T14) and tyrosine 15 (Y15). In fission yeast, the Wee1 and mitosis inhibitory kinase 1 (Mik1) protein kinases phosphorylate Y15 in Cdc2. WEE1Hu is the only known protein kinase that can carry out this inhibitory phosphorylation on Y15 in higher eukaryotes. In the present study, we examined the endogenous products of WEE1Hu in human cells and found that the original WEE1Hu cDNA lacked 214 amino acids at the N-terminus. The predicted full-length protein has weak, but significant, similarity over its entire length with Mik1. Thus, we suggest that 'WEE1Hu' is a Mik1-related protein rather than a Wee1 homologue. When isolated in immunoprecipitates, the endogenous WEE1Hu phosphorylated several cyclin-associated CDKs on Y15. WEE1Hu activity increased during S and G2 phases in parallel with the level of protein. Its activity decreased at M phase when WEE1Hu became transiently hyperphosphorylated. In addition, a decrease in WEE1Hu protein level was observed at M/G1 phase. Apparently, the hyperphosphorylation and degradation in combination caused inactivation of WEE1Hu at M phase and the following G1 phase. These results suggest that the activity of WEE1Hu is regulated by phosphorylation and proteolytic degradation, and that WEE1Hu plays a role in inhibiting mitosis before M phase by phosphorylating cyclin B1-Cdc2.     

Expansion of CD34+ cells from human umbilical cord blood by FL and/or TPO gene transfected human marrow stromal cell lines

To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34⁺ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34⁺ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34⁺ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3 ±52.1)-fold, total progenitor cells (CFC) by (74.5 ±5.2)-fold and CD34⁺ cells by 15.7-fold. Maximal expansions of CFC and CD34⁺ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 ± 5.5)-fold and (57.0 ± 19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34⁺ cells within 28 days was found only in two combinations, i.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant difference between these two groups statistically. These results suggest that human umbilical cord blood CD34⁺ cells can be extensively expandedex vivo by using gene transfected stromal cells along with cytokines.     

Analyses to clarify rich fractions in hepatic progenitor cells from human umbilical cord blood and cell fusion

Umbilical cord blood (UCB) is a source of hematopoietic stem cells and other stem cells, and human UCB cells have been reported to contain transplantable hepatic progenitor cells. However, the fractions of UCB cells in which hepatic progenitor cells are rich remain to be clarified. In the present study, first, the fractionated cells by CD34, CD38, and c-kit were transplanted via portal vein of NOD/SCID mice, and albumin mRNA expression was examined in livers at 1 and 3 months posttransplantation. At 1 and 3 months, albumin mRNA expression in CD34+UCB cells-transplanted livers was higher than that in CD34- cells-transplanted livers. Albumin mRNA expression in CD34+CD38+ cells-transplanted livers was higher than that in CD34+CD38- cells-transplanted [corrected] liver at 1 month. However, it was much higher [corrected] in CD34+CD38- cell-transplanted livers at 3 months. Similar expression of albumin mRNA was obtained between CD34+CD38+c-kit+ cells- and CD34+CD38-c-kit- cells-transplanted livers, and between CD34+CD38-c-kit+ cells- and CD34+CD38-c-kit- cells-transplanted livers, respectively. Second, fluorescence in situ hybridization and immunohistochemistry were performed to examine whether UCB cells really transdifferentiated into hepatocytes or they only fused with mouse hepatocytes. In mouse liver sections, of 1.2% cells which had human chromosomes, 0.9% cells were due to cell fusion, whereas 0.3% cells were transdifferentiated into human hepatocytes. These results suggest that CD34+UCB cells are rich fractions in hepatic progenitor cells, and that transdifferentiation from UCB cells into hepatocytes as well as cell fusion simultaneously occur in this situation.     

逆转录病毒载体介导MGMT和MDR1基因增强人脐血CD34＋细胞对联合化疗抗性的研究

To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O6-methylguanine-DNA-methyltransferase (MGMT) and multidrug resistance gene (MDR1) increase resistance to 1,3-Bis(2-Chloroethy1)-1-Nitrosourea (BCNU) and P-glycoprotein effluxed drugs, the present authors obtained a full length cDNA fragment encoding MGMT from liver tissue of a patient with cholelithiasis by RT-PCR. A bicistronic retroviral vector G1Na-MGMT-IRES-MDR1 cDNA was constructed and transfected the packaging cell lines GP + E86 and PA317 by electric performation method, using the medium containing VCR and BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, cord blood CD34+ cells were enriched with a high-gradient magnetic cell sorting system (MACS), and then transfected repeatedly with supernatant of retrovirus containing human MGMT and MDR1cDNA under stimulation of hemapoietic growth factors. PCR, RT-PCR, Southern blot, Northern blot, Western blot, FACS and MTT assay were used to evaluate the transfer and expression of the double genes in cord blood CD34+ cells. The cDNA encoding MGMT was verified by DNA sequencing and the bicistronic retroviral vector was confirmed by restriction endonuclease analysis. The purity of cord blood CD34+ cells was approximately 92% and recover rate was 75%, the highest titer of recombinant amphotropic retrovirus in the supernatant was up to 5.8 x 10(5) cfu/ml. The efficiency of gene transduction was 18% and 20% tested by colony formation and PCR, respectively. No helper virus was found by both nested PCR and rescue assay. The results showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The MTT analysis showed a 4.5 to 7.8-fold increase of resistance of transducted cells to BCNU and P-glycoprotein effluxed drug as compared with the nontransduced cells. This study provided a foundation for ameliorating combination chemotherapy toxicity in tumor clinical trial.     

Stem cell factor and FLT3-ligand are strictly required to sustain the long-term expansion of primitive CD34+DR- dendritic cell precursors

We studied cytokine-driven differentiation of primitive human CD34(+)HLA-DR(-) cells to myeloid dendritic cells (DC). Hemopoietic cells were grown in long-term cultures in the presence of various combinations of early acting cytokines such as FLT3-ligand (FLT3-L) and stem cell factor (SCF) and the differentiating growth factors GM-CSF and TNF-alpha. Two weeks of incubation with GM-CSF and TNF-alpha generated fully functional DC. However, clonogenic assays demonstrated that CFU-DC did not survive beyond 1 wk in liquid culture regardless of whether FLT3-L and/or SCF were added. FLT3-L or SCF alone did not support DC maturation. However, the combination of the two early acting cytokines allowed a 100-fold expansion of CFU-DC for >1 month. Phenotypic analysis demonstrated the differentiation of CD34(+)DR(-) cells into CD34(-)CD33(+)DR(+)CD14(+) cells, which were intermediate progenitors capable of differentiating into functionally active DC upon further incubation with GM-CSF and TNF-alpha. As expected, GM-CSF and TNF-alpha generated DC from committed CD34(+)DR(+) cells. However, only SCF, with or without FLT3-L, induced the expansion of DC precursors for >4 wk, as documented by secondary clonogenic assays. This demonstrates that although GM-CSF and TNF-alpha do not require additional cytokines to generate DC from primitive human CD34(+)DR(-) progenitor cells, they do force terminal differentiation of DC precursors. Conversely, FLT3-L and SCF do not directly affect DC differentiation, but instead sustain the long-term expansion of CFU-DC, which can be induced to produce mature DC by GM-CSF and TNF-alpha.     

IL-3 increases production of B lymphoid progenitors from human CD34+CD38- cells

The effect of IL-3 on the B lymphoid potential of human hemopoietic stem cells is controversial. Murine studies suggest that B cell differentiation from uncommitted progenitors is completely prevented after short-term exposure to IL-3. We studied B lymphopoiesis after IL-3 stimulation of uncommitted human CD34+CD38- cells, using the stromal cell line S17 to assay the B lymphoid potential of stimulated cells. In contrast to the murine studies, production of CD19+ B cells from human CD34+CD38- cells was significantly increased by a 3-day exposure to IL-3 (p < 0.001). IL-3, however, did not increase B lymphopoiesis from more mature progenitors (CD34+CD38+ cells) or from committed CD34-CD19+ B cells. B cell production was increased whether CD34+CD38- cells were stimulated with IL-3 during cocultivation on S17 stroma, on fibronectin, or in suspension. IL-3Ralpha expression was studied in CD34+ populations by RT-PCR and FACS. High IL-3Ralpha protein expression was largely restricted to myeloid progenitors. CD34+CD38- cells had low to undetectable levels of IL-3Ralpha by FACS. IL-3-responsive B lymphopoiesis was specifically found in CD34+ cells with low or undetectable IL-3Ralpha protein expression. IL-3 acted directly on progenitor cells; single cell analysis showed that short-term exposure of CD34+CD38- cells to IL-3 increased the subsequent cloning efficiency of B lymphoid and B lymphomyeloid progenitors. We conclude that short-term exposure to IL-3 significantly increases human B cell production by inducing proliferation and/or maintaining the survival of primitive human progenitors with B lymphoid potential.