Extended Culture of Bone Marrow with Granulocyte Macrophage-Colony Stimulating Factor Generates Immunosuppressive Cells

Bone marrow-derived dendritic cells (BM-DCs) are generated from bone marrow (BM) cells cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) for a week. In this study we investigated the effect of duration on the BM culture with GM-CSF. Within several months, the cells in the BM culture gradually expressed homogeneous levels of CD11c and major histocompatibility complex II on surface, and they became unable to stimulate allogeneic naïve T cells in mixed lymphocyte reaction (MLR). In addition, when the BM culture were sustained for 32 wk or longer, the BM cells acquired ability to suppress the proliferation of allogeneic T cells in MLR as well as the response of ovalbumin-specific OT-I transgenic T cells in antigen-dependent manner. We found that, except for programmed death-ligand 1, most cell surface molecules were expressed lower in the BM cells cultured with GM-CSF for the extended duration. These results indicate that BM cells in the extended culture with GM-CSF undergo 2 distinct steps of functional change; first, they lose the immunostimulatory capacity; and next, they gain the immunosuppressive ability.

Genotoxicity Evaluation of Dipotassium -Trioxohydroxytetrafluorotriborate, K2(B3O3F4OH), in Human Lymphocyte Cultures and Mice Reticulocytes

ABSTRACT Genotoxic effects of inorganic molecule dipotassium-trioxohydroxytetrafluorotriborate, K2(B3O3F4OH), a promising new therapeutic for the epidermal changes treatment, have been evaluated. In vitro analysis included evaluation of genotoxic and cytotoxic potential of K2(B3O3F4OH) in concentrations of 0.01, 0.02, 0.05 and 0.06 mg/mL applying cytokinesis-block micronucleus cytome assay in human lymphocyte culture. With the increase of concentration the frequency of micronuclei elevated but the differences were not significant. Also, there were no significant differences among the frequencies of nuclear buds and nucleoplasmic bridges between controls and treated cultures. Nuclear division index and nuclear division cytotoxycity index values did not reveal significant cytotoxic effect of K2(B3O3F4OH). In vivo genotoxic effects were analyzed on BALB/c mice applying reticulocytes micronucleus assay. K2(B3O3F4OH) was administrated intraperitoneally in final concentrations of 10, 20, 50 and 55 mg/kg. Significant decrease of reticulocytes ratio and increase of micronuclei frequencies against pre-treatments were found for both sampling periods of 48 and 72 hours of the highest applied concentration. This study confirmed that K2(B3O3F4OH) is not genotoxic in tested concentrations in vitro as well as in concentrations lower than 55 mg/kg in vivo. This study presents a reliable basis for further pre-clinical and potential clinical investigations.

The function of murine immature CD8α+ dendritic cells in vitro / 中华器官移植杂志

Objective To observe the function of immature CD8α+ dentritic cells (DCs) in vitro. Methods The bone marrow and spleen of C57BL/6(H-2b) and Balb/c (H-2d) mice were got to prepare immature CD8α+ DCs and spleen lymphocytes,and treated by mytomycin. MTT test was used.MLR group, MLR plus variable density syngeneic CD8α+ DC group, MLR plus variable density allogeneic CD8α+ DC group,MLR plus variable density CD8α+ DC supernatant group,CD8α+ DC plus syngeneic T cell group and negative control group were established. MLR group was set up by responder cell ratio of 0.2,0.5,0.8,1.0,to build the MLR plus syngeneic and allogeneic CD8α+ DC experimental groups. Culture supernatant from different density (1 × 105/ml - 5 × 106/ml) of CD8α+DCs was added into MLR to build CD8α+ DC supernatant group. CD8α+ DCs were co-cultured with syngeneic T cells to build CD8α+ DCs plus syngeneic T cells group. 2 × 105/well responder cells served as the negative control group. ELISA was used to detect the concentrations of IFN-γ and IL-10 in the DCs could both suppress MLR (P＜0. 05), and the difference was not statistically significant (P＞0. 05). When CD8α+ DCs were increased, the suppressive effect was enhanced. When CD8α+ DC/responder cell ratio ＞0. 2, the inhibitory effect could be observed, and this effect reached the peak when the ratio was 1.0. The CD8α+ DCs had weak ability to stimulate syngeneic lymphocyte proliferation in vitro, and certain stimulating effect could be seen only when CD8α+ DC/responder cell ratio ＞2 (P＜0. 05). Its culture supernatant also showed suppressive effect (P＜0. 05), and the supernatant with a cell density of 5 × 105/ml showed the maximum effect. IL-10 concentration in the concentration was 1.0 ± 1.2 pg/ml. Conclusion The in vitro function of immature CD8α+ DCs was immunosuppression/tolerance,and they could secret high level of IL-10. The CD8α+ DCs and their culture supernatant could suppress MLR in vitro.

Donor dendritic cells-mediated recipient lymphocyte reaction after living related kidney transplantation / 中华器官移植杂志

Objective To explore the feasibility of mediating recipient lymphocyte reaction with donor dendritic cells (DCs) in renal allograft recipients. Methods Donor bone marrow monocytes (BMMCs) were isolated and cryopreserved in liquid nitrogen before kidney transplantation. At 0 day, 1month,3 month, 6 month and 9 month post-operation, CD34+ cells which were isolated from frozen BMMCs and cultured into DCs as well as the peripheral blood lymphocytes (PBLs) of donors were used as the stimulating cells to the PBLs of recipients and healthy volunteers. The number of viable DCs from frozen- and room temperature-preserved BMMCs was counted and the reactions of recipients'and healthy volunteers' lymphocytes to DCs and donor PBLs were measured. Results 6. 8 × 107BMMCs were isolated from each 10 ml of donor bone marrow on average while (4. 10 ± 0. 58) × 105CD34+ cells were isolated by magnetic active cell sorting (MACS). There was no significant difference in the isolating rate of recovered CD34+ cells at each observation point postoperatively. The percentage of viable BMMCs and CD34+ was decreased significantly at 1 month after surgery, then, decreased slowly and progressively. The decreasing rate of BMMCs was higher than CD34+. The rate of viable DCs was maintained stable (93. 2%-94. 8% ) in each group. The reactions of recipients' and healthy volunteers' lymphocytes to DCs were stronger than those to donor PBLs (P＜0. 05). The reactions of healthy volunteers' lymphocytes to DCs were maintained stable while those of recipients' were fluctuating. Conclusion Bone marrow-derived DCs are superior to PBLs in mediating long-term lymphocyte reaction after kidney transplantation due to their stable viability and stimulating ability to lymphocytes. Only once collection of a small quality of bone marrow of donors is needed to meet the demand of immune monitoring at any time after transplantation.

Role and mechanism of human Kupffer cells in liver allo-geneic transplantation / 中华肝胆外科杂志

Objective To explore the possible role and mechanism of the Kupffer cells (KCs) in liver allo-geneic transplantation at the early stage. Methods In vitro cell contact coculture system was established. Culture supernatants were collected respectively on the 1st, 2nd, 4th, 6th d after cocul-ture and the KCs and PBMCs were harvested on the 6th day after culture. The expression of HLA-G on the membrane of the KCs and PBMCs was detected with immunochemistry. Nitrate reduction test was used to determine the concentration of nitric oxide. IFN-γ, IL-10, TGF-β1 cytokine levels in the supernatants were also measured with ELISA. The proliferation of lymphocytes was evaluated with MTT. Results six days later, no HLA-G molecules were detected on the membrane of the KCs and PBMCs. In the experimental group containing KCs, the levels of NO, IL-10 and TGF-β1 was signifi-cantly increased(P<0. 05), while the levels of IFN-γ was relatively lower(P<0. 05) as compared to the experimental group without KCs. No IL-10 and IFN-γ were detected in the control group, and on-ly few NO and TGF-β1 was found in the control group with KCs. MTT test showed that the value of optical density was lower in the experimental group with KCs than that in any other group(P<0. 05).Conclusion No HLA-G is expressed on the membrane of KCs and PBMCs after contact coculture.KCs may participate in regulating production of NO and Th2/Th3-like cytokines and suppressing the proliferation of lymphocytes, through which KCs probably take part in inducing immunotolerance of liver transplantation in early stage.

Establishment of in vitro stable culture of human peripheral blood dendritic cells and its comparation with magnetic activated cell sorting / 中国地方病学杂志

Objective To establish a economic and stable method to induce and culture dendritic cells (DCs) from peripheral blood of human being, and compare with the magnetic activated cell sorting. Methods Monocytes were isolated from health donors peripheral blood mononuclear cells(PBMC) by density gradient separation,cultured and compared with that of cells isolated by the magnetic activated cell sorting or adherent culture,respectively. PBMC were cultured with recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) and recombinant human interleukin-4(rhIL-4) for 6 days to induce the growth of DCs. Morphological changes was observed under inverted microscope. Meanwhile, cell viability was tested at the 3rd, 5th, 6th day,respectively. The phenotypes, like CD14, CDla, HLA-DR were analyzed with flow cytometry after PBMC were adherent cultured for 1, 2, 5 h. After adding human recombinant cytokines, the phenotypes of acquired cells surface markers, CD14, CD1a, CD86, CD83 and HLA-DR would be detected and compared with flow cytometry. T cells proliferating activity was determined by allogeneic mixed lymphocyte reaction in vitro. Results After adherent culture for 2 h, the acquired DCs showed typical morphology. Cell viability was decreased at days 5th, 6th[(53.333 ±5.774)%,(38.333 ± 7.638)%] than that at day of 3rd[(68.667 ± 3.215)%, all P ＜ 0.05] with the magnetic activated cell sorting, but with adherent culture method, the difference was not statistically significant (F = 0.737,P＞ 0.05) at days of 3rd, 5th, 6th[(92.667 ± 3.055)%,(94.000 ± 1.000)%,(94.667 ± 1.528)%]. Moreover,compared with the magnetic activated cell sorting, there were differences in cell viability of adherent culture method at days of 3rd, 5th, 6th(t = 9.374, 12.021,12.527, all P ＜ 0.05). Before and after using the magnetic activated cell sorting, the expression of CD14 were (32.457 ± 12.351) %, (41.914 ± 14.858)%, respectively. The difference was not statistically significant(t = 1.295, P ＞ 0.05). After culturing for 2 h, the expression of CD14[(35.267 ± 4.658)%]was higher than those of culturing for 1, 5 h[(15.033 ± 6.189)%, (21.233 ± 4.895)%, all P ＜ 0.05]. Compared with the 1st day[(32.328 ± 14.517)%], the CD14 expression level[(2.200 ± 1.356)%] on surface of DCs was significantly reduced(t = 5.467, P ＜ 0.05) at the 6th day of culturing, the CD1a expression level[(43.371 ±16.250)%] was remarkablely increased than that of the 1st day[(12.300 ± 6.223)%, t = 2.545, P ＜ 0.05];while the expressions of CD86, CD83, HLA-DR[(16.857 ± 5.686)%,(9.343 ± 5.230)%,(72.800 ± 17.881)%] were similar(t = 0.652,1.137,0.907, all P ＞ 0.05) compared with that of the 1st day[(12.550 ± 16.758)%, (6.250 ±1.323)%, (64.671 ± 15.588)%]. In mixed lymphocytes reactions, with increasing of lymphocytes, T lymphocytes proliferating activities were reduced. In the magnetic activated cell sorting, when the ratio of DCs and lymphocytes were 1: 50, 1: 100, cells proliferation ability(1.502 ± 0.055,1.507 ± 0.029) were lower than that of ratio of 1: 10(1.859 ± 0.049, all P ＜ 0.05);in adherent culture method, the ratio of DCs and lymphocytes was 1: 100, the cells proliferation ability(1.545 ± 0.066) was decreased than that of ratio 1: 10(2.015 ± 0.301, P ＜ 0.05). When the proportion of DCs and lymphocytes remained the same, the capacity to stimulate T lymphocyte was similar of the two methods(P ＞ 0.05). Conclusions Comparied with the magnetic activated cell sorting, after culture of PBMC for 2 h the induction of DCs can produce better formed and functional cells, and this method is stable, simple,economic, and is a suitable method for basic and clinical research of DCs in vitro.

Experimental study on induction of allo-hyporesponsiveness by ICOS-Ig in vitro and in vivo ZHANG / 中华器官移植杂志

Objective To express human inducible eostimulator (ICOS) extracellular region and IgG Fc fusion protein, and analyze their function in allogenie lymphocyte proliferation in vitro and in vivo. Methods Human ICOS extraeellular region and IgG Fc fragment were cloned into a soluble expression vector. ICOS-Ig fusion protein was expressed and purified in CHO cells. To monitor primary MLR, Balb/c spleen T cells were isolated as responder cells, and irradiated C57BL/6 spleen cells as stimulator cells. 50 μg/ml ICOS-Ig or IgG was added to primary MLR cultures. The cells responsive rates were detected by 3 H-TdR methods. ELISA tested supernatants for eytokines (IL-2,IL-4, IL-10 and IFN-γ). T cells of each group in primary MLR were cultured as responder cells for secondary MLR, and irradiated C57BL/6 (donor) or C3H (third party) spleen cells as stimulator cells. Similar indexes were detected in secondary MLR. Then vital dye CFSE was used to study alloreactive T cell proliferation in vivo. CFSE-labeled C57BL/6 spleen cells were transferred to irradiated Balb/c mice. Mice were then intraperitoneally injected with 0. 2 mg IgG, ICOS-Ig or CsA each day.At the 3rd day after transfection, the spleen cells of the mice were harvested to detect CD4+ CFSE+ and CD8+ CFSE+ by FACS. Results In primary MLR, ICOS-Ig inhibited allogenic T-cell proliferation with inhibition rate being (58 ± 8)% in 50 μg/ml, and increased IFN-γ secretion. In secondary MLR, ICOS-Ig specifically inhibited the proliferation of donor spleen cells with inhibition rate being (42±8)%, and in ICOS-Ig group the levels of IL-4 and IL-10 were lower and the level of IFN-γ higher than in IgG group. However, ICOS-Ig didn't inhibit the proliferation of third-party spleen cells. In the CFSE dye assay, CFSE intensity of CD4+ and CD8+ T cells in ICOS-Ig and CsA groups was stronger than that in control group (P < 0. 05), while CFSE intensity in combined treatment group were even stronger than that in ICOS-Ig and CsA groups (P<0. 05). Conclusion ICOS-Ig could inhibit allo-reactive T cell proliferation in vitro and in vivo, and induce donor-specific T cell hyporesponsiveness specifically.

In vitro induction of allo-T lymphocytes proliferation by myeloid dendritic cells in patients with severe aplastic anemia / 中华内科杂志

Objective To investigate the function of myeloid dendritic cells (mDCs) from severe aplastic anemia ( SAA ) patients in stimulating allogeneic T lymphocytes proliferation in vitro and then explore the immunopathogenesis of SAA. Methods Twenty-five SAA patients ( 15 untreated and 10 recovered after immunosuppressive therapy) and 12 normal controls were enrolled in this study. Their mature mDCs were induced from their bone marrow monocytes with recombined human interleukin-4 ( rhIL-4) , recombined human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombined human tumor necrosis factor (rhTNF) in vitro. Then mDCs were co-cultured with allogeneic lymphocytes (mixture lymphocyte reaction, MLR) at a ratio of 1: 100 or 1: 50. The growth rate of lymphocyte was measured with methyl thiazolyl tetrazolium ( MTT) colorimetry.The concentrations of interleukin( IL) -12 and inlerferon -y (IFNγ) in MLR supernatant were measured with EL1SA. The correlation between the growth rate and the concentration of IL-12 or IFNγ was analyzed. Results When mDCs and lymphocytes were co-cultured at the ratio of 1: 100, the growth rates of lymphocytes stimulated with mDCs from untreated, recovered SAA patients and controls were (219. 8 ±94. 0)% , (159. 1 ±66. 0)% and (160. 1 ±91. 9)% respectively. The concentrations of IL-12 in MLR supernatant were (8. 2 ± 3. 6) ng/L, (6. 5 ± 2. 8) ng/L and (6. 1 ± 2. 6) ng/L and the concentrations of IFNγ were (21. 8 ± 8. 7) ng/L, (25. 5 ± 9. 1) ng/L and (22. 6 ± 7. 8) ng/L respectively. All of them had no statistical differences among the three groups ( P > 0. 05 ). When mDCs and lymphocytes were co-cultured at the ratio of 1: 50, the growth rate of lymphocytes stimulated with mDCs from untreated patients was (322. 1 ± 171. 1)% , which was higher than that of recovered patients [ (180. 9 ±79. 1)% and controls (192. 3 ±91. 9)% ]. The concentrations of IL-12 in MLR supernatant in the three groups were (12.6 ±4.4) ng/L, (9.4 ±3.3) ng/L and (8.5 ±3.7) ng/L, and the concentrations of IFNγ were (32. 3 + 9. 2 ) ng/L, ( 27. 4 ± 6. 5) ng/L and (24. 4 ± 7. 4 ) ng/L Both of the values in untreated cases were higher than those of the recovered cases or controls (P < 0. 05 ) , but there were no statistical difference between the recovered and control groups ( P >0. 05 ). The concentration of IL-12 in MLR supernatant correlated positively with the growth rate of lymphocyte (r=0. 529,P <0. 01) and so did the concentration of IFNγ (r = 0. 381, P < 0. 05). Conclusion The function of mDCs to stimulate T lymphocytes proliferation in SAA was enhanced; it might play an important role in the immunopathogenesis of SAA.

Prolongation of allograft survival by donor mesenchymal stem cells infusion in rat heart transplantation / 中华器官移植杂志

Objective To investigate the immunomodulatory effect of mesenchymal stem cells (MSCs) and their role in prolonging allograft survival in rat heart transplantation. Methods Inbred Wistar rats were used as donors, and Fisher 344 as recipients. MSC were isolated from femur and tibia bone marrow of donors and cultured in vitro. Mixed lymphocyte reaction assays were performed to assess the immunosuppressive effects of different concentrations of MSC on allogeneic T cell proliferation. Cardiac allograft model was established and according to different intervention measures recipients were divided into two groups (MSC treatment group and control group) (n=8 in each group). In MSC treatment group, recipients were infused with 2×106 MSC via the tail vein at designated intervals (one week before operation, during operation and consecutive three days postoperation), while in control group, the recipients were treated with Ringer's solution at the same interval& At day 5 posttransplantation real-time PCR was used to detect the changes in the expression of Thl and Th2 cytokine genes in transplanted hearts. Results In vitro allogeneic T cell response was greatly suppressed by MSC in a dose-dependent manner. Real-time PCR revealed that IL-1β,IFN-γ, IL-4 and IL-10 were expressed in MSC treatment group, while IL-4 and IL-10 were not expressed in control group but with significantly higher expression of IL-1β and IFN-γ. As compared with control group, survival of MSC-treated allografts was markedly prolonged as compared with control group (mean survivaldays: 12.4±5.3 vs 6.4±2.0, P＜0.05). Conclusion Intravenous adrninistmtion of MSC can prolong the survival of transplanted heart possibly by induction of allograft tolerance through changing Th1/Th2 balance.

The alteration of CD4+ regulatory T cells in patients with ankylosing spondylitis / 中华微生物学和免疫学杂志

Objective To evaluate the alteration of CD4+ regulatory T cells in peripheral blood from patients with ankylosing spondylitis(AS). Methods Seventy-eight AS patients and 50 healthy individuals were included in this study. The proportion of CD4+CD25+CD127lo/- T cell population in CD4+ T cells as well as that cytotoxic T lymphocytes(CTL) and NK cells in lymphocytes was evaluated by flow cytometry. Serum TGF-β and TNF-α were measured by ELISA. The inhibitory function of CD4+CD25+ regulatory T cells was measured by mixed lymphocyte culture. Results The population of CD4+CD25+CD127lo/- T cells in peripheral blood of AS patients accounted for (4.36±1.21)% of CD4+ T lymphocytes, which was significantly lower than that in healthy individuals (P<0.05). The CD4+CD25+CD127lo/- T cells population in AS patients was positively correlated with TGF-β level, but negatively with TNF-α. Compared with healthy individuals, the function of CD4+CD25+ regulatory T cells in the inhibition of alloreactive T cells was lower in AS patients, which was related to the decreased secretion of TGF-β. Conclusion The CD4+ regulatory T cells in peripheral blood of AS patients are significantly decreased and its function is defective, which leads to immune regulatory dysfunction in vivo. It may be one of immune pathogenesis mechanisms of AS.

Vector construction and expression of soluble mPDL1-hIgGFc and its effect on the proliferation and apoptosis of cells in vitro / 中华微生物学和免疫学杂志

Objective To construct vector expressing soluble mPDL1-hIgGFc and study its effect on the proliferation and apeptosis of cells in vitro. Methods The extrncellular domain of mPDL1 gene was amplified from pmPDL1 vector by PCR and inserted into phIgGFc vector. The recombinant pmPDL1-hIgGFc was transfected into CHO cells by LipofectAMINETM2000, and the transfected cells were named as CHOp. The expression of mPDL1-hIgGFc in the culture supernatants of CHOp was assayed by ELISA and Western blot. The effects of CHOp culture supernatants on mixed lymphocyte culture(MLC) was analysed by Flowm-etry. Results The extracellular domain of mPDL1 gene were obtained from PCR. DNA sequencing and the identification of digestion by HindⅢ and KpnⅠ indicated the recombinant plasmid pmPDL1-hIgGFc was suc-cessfully constructed. ELISA and Western blot analysis proved that the CHOp could express mPDL1-hIgG-Fc. CHOp culture supernatants could inhibit lymphocyte proliferation and induce the apoptosis of the activa-ted T cells in MLC in vitro in a dose-dependent manner. Conclusion The mPDL1-hIgGFc protein could in-hibit lymphocyte proliferation and induce the apoptosis of the activated T cells.

Effect of mesenchymal stem cell on the rate of Th1/Th2 with different stimulations / 基础医学与临床

Objective To investigate the effect of mesenchymal stem cell(MSC) on the balance of Th1/ Th2 with different stimulations.Methods To inoculate MSC in 24-well tissue culture plates.after 3 days, added T lymphocytes co-stimulated by PMA and Ionomycin,or the MSC were added to the two-way mixed lymphocyte culture according to different proportion.The subsets and cytokines of T lymphocytes were analyzed by flow cytomety.Results Differentiation into Th1 of T lymphocytes activated by co-stimulation can be inhibited MSC;In MLC,CD8~+T cell subsets and Th1 were evidently decreased.But Th2 cells were slightly increased.Conclusion MSC can significantly suppress CD8~(+)T cell,Th1,increase Th2.MSC has potentialities of alleviating acute graft-versus-host disease(aGVHD) and maintaining graft versus leukemia(GVL).

Study of fetal lymphocyte of intrahepatic cholestasis of pregnancy / 中华妇产科杂志

Objective To explore effect of fetal lymphocyte on pathogenesis of intrahepatic cholestasis of pregnancy(ICP).Methods Twenty pregnant women with ICP and 20 normal pregnant women were enrolled in the study.The single mixed lymphocyte culture/reaction(MLC/MLR)was conducted using inactive lymphocyte obtained from maternal peripheral blood and lymphocyte of cord blood from fetus.Antigen-induced-lymphocyte-proliferation-reaction was used for dermic soluble antigen and decidual soluble antigen obtained from maternal blood and cord blood from fetus.The intense of proliferation was calculated and compared between normal and ICP-complicated pregnancies.Results(1)The level of intense of proliferation of fetal lymphocyte was significantly increased in ICP group 2.75?0.36 than those of normal control group 1.45?0.19 in single mixed lymphocyte culture(P＜0.05).(2)The level of intense of proliferation of fetal lymphocyte was significantly increased in ICP group 1.45?0.19 than those of normal control group 0.67?0.24 in decidual soluble antigen induced lymphocyte proliferation reaction(P＜0.05). (3)The level of intense of proliferation of fetal lymphocyte was significantly increased in ICP group(1.22?0.44)than those of normal control group(0.66?0.27)in dermic soluble antigen induced lymphocyte proliferation reaction.Conclusions(1)The fetal lymphocyte may be one of the effector cells in pathogenesis of ICP.(2)The disturbance of fatal-maternal immune-tolerance is one of the important mechanisms underlying ICP.

Immunosuppressive action of Stro-1~+ and Stro-1~- subgroups of human mesenchymal stem cells / 解放军医学杂志

Objective To explore the difference of immunosuppresive effect between the expanded Stro-1+ and Stro-1-subgroups of human mesenchymal stem cells in vitro. Methods Mononuclear cells (MNCs) were isolated from bone marrow (BM) samples and seeded in a T-75 cm2 tissue culture flask contained with Dexter medium. When 50% confluence was obtained, adherent cells were collected and incubated with anti-stro-1 antibody, and the Stro-1+ and Stro-1-MSCs were further seeded for expansion. The total culture time (median) was 15 days. Cells were then analyzed by flow cytometry. One-way mix lymphocyte reaction (MLR) (1?105 responding cells and an equal number of stimulating cells/well were co-cultured in 96-well plates) and nonspecific mitogenic stimuli phytohemagglutinin (PHA) plus interleukin-2 (IL-2) induced lymphocytes proliferation (PBL 1?105/well were mixed with PHA 10?g/ml and IL-2 500U/ml in 96-well culture plates) were established in vitro. 1?103-3?104 irradiated Stro-1+ MSCs and Stro-1-MSCs were added into the two systems at the beginning of reaction. Immunosuppressive actions of both Stro-1+ or Stro-1-MSCs were compared. Results Adherent cells contained a median of 9% (range 5%-26%) Stro-1+ cells, which expressed higher immunophenotype of MSCs. In both reaction systems, suppressive actions occurred in a dose-dependent fashion when whatever Stro-1+ MSC or Stro-1-MSC were added. However, that the addition of 1?103 Stro-1-cells enhanced rather than inhibited the lymphocyte proliferation in one-way MLR. In the presence of various concentrations of MSCs, Stro-1+ MSCs always showed a significantly increased inhibitory effects in comparison to Stro-1-MSCs (P

Immunophenotypes and Proliferative Function of Lymphocytes in Umbilical Cord Blood / 대한임상병리학회지

BACKGROUND: The umbilical cord blood (UCB) is highlighted to be a alternative source of hematopoietic stem cells with a hope of resolving major problems of bone marrow transplantation, which are the lack of suitable HLA-matched donors and the occurrence of graft-versus-host disease (GVHD). In this study we have assessed the immunological potential of lymphocytes in UCB, which could mediate GVDH, by immunophenotypes and functional assays on 20 UCB and 19 adult peripheral blood as controls. METHODS: Immunophenotyping of lymphocytes were performed by one and two color flow cytometry (FACSort, Becton Dickinson, USA) using monoclonal antibodies (CD3, CD4, CD8, CD19, CD16/56, CD45RA, CD45RO, HLA-DR, HLA-DQ, HLA-DP, TCRalphabeta, TCRgammadelta). To assess proliferative responses, UCB lymphocytes were cultured in the presence of phytohemagglutinin (PHA) (5, 20, 100 ug/mL) and concanavalin A (ConA) (1, 5, 10 ug/mL). After 72 hour culture and 3H-thymidine pulsing, specific 3H-thymidine uptake was compared to control cells (without mitogen) by stimulation index (SI). RESULTS: In phenotypic analyses, the percentages of CD4, CD8, CD45RO, TCRgammadelta and HLA- DP positive cells were significantly lower in UCB compared with the adult controls, while the percentage of CD45RA positive cells was increased in UCB. In addition, the percentages of B cell (CD19), NK cell (CD16/56), HLA-DR, HLA-DQ and TCRalphabeta were comparable to those in adult controls. In mitogen stimulated culture, UCB lymphocytes showed a significant lower proliferative responses at 20, 100 ug/mL of PHA, and 5, 10 ug/mL of ConA concentrations. CONCLUSIONS: We have observed that UCB lymphocytes appeared to be phenotypically and functionally immature. Thus, UCB might be a attractive source for hematopoietic stem cells in that these UCB cells may have lower immunological potentials mediating GVHD after transplantation.

Mechanism of immunosuppressive effects of triptolide / 中国药理学通报

Triptolide (TL) is a major active component extracted from Chinese traditional herb Tripteryginm wil-fordii Hook. In this article, the effects of TL on mixed lymphocyte culture (MLC),suppressor T cell(Ts) activity, delayed type hypersensitivity (DTH) reaction, IL - 2 secretion activity and Th/Ts ratio were evaluated, In vitro TL 0. 5~10 ?g ? L-1suppressed one-way MLC,Lymphocytes induced in first MLC with TL of 5,10 ?g ? L-1 suppressed the second MLC after irradiation with 3000 rad 60Co source. This suggests that TL may induce Ts cell-s. In vivo, DTH reaction sensitized to dinitrofluo-robenzene (DNFB) monitored by the increase in weight of the ears challenged with antigen was suppressed by TL at doses of 0. 12 to 0. 50 mg ? kg-1(ip,qd?5) and the IL -2 activity secreted by spleen cells of these mice was inhibited at doses of 0. 25 and 0. 5 mg ? kg-1. TL 0. 25, 0. 5 mg ? kg-1(ip,qd?8) also had prominent suppres-sive effects on enhanced DTH reaction which was induced by cyclophosphamide(250 mg ? kg-1,ip). Th/Ts ratio of mouse thymus cells were reduced after given 0. 25 mg ? kg-1 of TL for 5 consecutive days. The above-mentioned data suggest that TL has suppressive effects on cellular immunity function and the suppressive mechanism may be related to the suppression of Th cells and IL-2 secretion activity and the induction of Ts cells.

Effects of recombinant immunotoxin hIL-2-LuffinP1 on lymphocyte proliferation in vitro / 解放军医学杂志

Objective To observe the biological activity of the recombinant immunotoxin hIL-2-LuffinP1 derived by gene engineering in vitro.Methods To observe the inhibitory effect on lymphocytes in vitro,the splenocytes isolated from C57 and BALB/c mice were used for mixed mouse lymphocyte response(MLR),and the lymphocyte proliferation was observed in vitro with BALB/c mice splenocytes stimulated by concanavalin A(ConA).Different concentrations of hIL-2-LuffinP1 was added into the reaction system,with LuffinP1 adopted as control.3H-TdR incorporation assay was used to detect the effects of hIL-2-LuffinP1 on lymphocyte proliferation,and its effects on CTLL-2 cells(IL-2 dependent cells) were also observed.The experiments consisted of six groups(PBS,IL-2,LuffinP1,LuffinP1+IL-2,hIL-2-LuffinP1 and hIL-2-LuffinP1+IL-2).CTLL-2 cells were cultured for 12h after being treated with different agents,and then the cells were stained by trypan blue to estimate the dead cells in every 100 cells.Results It was showed that CPM declined correspondingly to the gradually increased concentration of hIL-2-LuffinP1,and cell proliferation was inhibited obviously.The inhibition rate was up to 97% at the concentration of 10-6mmol/L of hIL-2-LuffinP1.The activity of inhibiting lymphocyte proliferation was proportional to the dosage.On the contrary,no obvious inhibition to lymphocyte proliferation was found in the control group treated with LuffinP1,the inhibition ratio only reached to 14% with the same concentration of hIL-2-LuffinP1.It was also found that,when hIL-2-LuffinP1 and LuffinP1 in different concentrations added to spleen cells stimulated by ConA,hIL-2-LuffinP1 still exhibited strong ability of inhibition on the splenocyte proliferation,while LuffinP1 has no obvious inhibiting effect on the lymphocyte proliferation in the two reaction systems mentioned above.MLR showed the same results.Furthermore,in the experiment of cytotoxic effects of IL-2-LuffinP1 on CTLL-2 cells,the inhibition rates of hIL-2-LuffinP1+IL-2 group and hIL-2-LuffinP1 group were 29.6% and 47.4% respectively,the difference was significant compared with IL-2 group(P

STUDIES ON THE EFFICACY OF DIFFERENT INDUCERS FOR INDUCING IMMUNE INTERFERON FROM HUMAN TONSILUR LYMPHOCYTES / 第二军医大学学报

The efficacy of different inducers used to induce immune interferon (IFN-?)from ionsillar cells of onsillectomized normal children was sfudied.Tonsillar cells were made in suspension of 1?107 cells/ml treated with antibiotics,and cultured for 2-3 days and then supernants were assayed for IFN-r.The results show that cells of individual human tonsil could produce IFN-? spo ntaneously owing to the induction by pharyngeal normal flora.Esculentoside (Es) alone or .pokeweed mitogen (PWM) alone,their combinatian or with other inducers were capable of inducing IFN-?.Among them,PWM was more effective in inducing IFN-? than Es,and its efficacy was similar to that of PWM+ scoppolamine,but higher than that of PWM+C.parvum or PWM+Con A.Mixed lymphocyte culture (MLC) of human tonsils,with or without PWM or Es,could produce IFN-?,but slightly higher titer was obtained with MLC+PWM.The possible reasons for the difference of efficacy of various methods in inducing IFN-? from human tonsillar cells are discussed.