| Objective1 To investigate the immunosuppressive effect of bone marrow derived mesenchymal stem cells (MSCs) on T cell proliferation in vitro. To study the main mechanisms of MSC-mediated immunomodulation.2 To investigate the changes in MSC immunomodulatory activity in the presence of Interferon-gamma (IFN-y). To explore the synergistic effects of IFN-y in the immunomodulatory role of MSC.Methods1 Human MSCs were generated from bone marrow aspirates of healthy donors, recruited after informed consent. MSCs were obtained with density-gradient centrifugation or direct plating and cultured in 25cm2 flasks. MSCs at passage 3 to 5 were cryopreserved and used for futrher study.2 MSCs were cultured in the presence or absence of IFN-y (100ng/ml), the supernatants were collected for measurement of PGE2,HGF and TGF-β1 by ELISA kits. T lymphocytes proliferated under the stimulation of anti-human CD3 monoclonal antibody (mAb) and anti-human CD28 mAb.HGF and TGF-β1 were added respectively into the culture system, their effects on T cell proliferation were examined by BrdU ELISA kit.3 MSCs were cultured in the presence or absence of IFN-y (100ng/ml) for 48h and total RNA was extracted from cells and reverse-transcribed to cDNA. The cDNA was analysed for the expression of human indoleamine 2,3-dioxygenase (IDO) mRNA by semiquantitative RT-PCR. The expression levels were compared in these two conditions. Kynurenine (100μM) was added into the T lymphocytes culture system, its suppressive effects on T cell proliferation was examined by BrdU ELISA kit.4 Mononuclear cells (MNC) were extracted from peripheral blood from different healthy donors. MSC were cultured for 24h prior to MNC addition. After 96h of incubation, BrdU labeling solution was added to each well and incubated for a further 24h. The cell proliferation was tested by BrdU ELISA kit. Furthermore, recombinant human IFN-γ(100ng/ml) and anti-IFN-γmAb (5μg/ml) were added into the culture system. The T cell proliferation was tested in these different conditions.Results1 MSCs could be isolated from normal human bone marrow. MSC cells obtained with density-gradient centrifugation or direct plating had similar characteristics in cell morphology and proliferation kinetics. After the MNCs were inoculated in the plastic culture flask, the cells developed to an adherent fibroblast-like population. Two to there weeks later, the cells could be digested with trpysin and expanded. By the third to fourth passage,3～5×106 MSC per donor could be retrieved for further experiments.2 MSCs from three different donos were inoculated in flat-bottomed 24-well plates (1×105/well). The supernatant of the culture media was collected every 24h. The immunosuppressive cytokines PGE2,HGF and TGF—β1 coule be detected within 24～48h. Concentrarions of these cytokines continually increased until 144h after inoculation.Furthmore, MSCs were divided into two groups, in experimental group, MSC was cocultured with IFN—γ(100ng/ml) and in control group, MSC was cultured alone. After 144h, the supernatant was collected for examination. In the experimental group, the concentrations of PGE2,HGF and TGF-β1 were 1715.5167±628.5726 pg/ml,4031.7733±1496.8027pg/ml and 1753.5363±413.8059 pg/ml respectively. In the control group, the concentrations of PGE2,HGF and TGF-β1 were 1344.5163±709.3583 pg/ml,2452.4267±1375.3291 pg/ml and 1026.6080±450.5418 pg/ml respectively. Therefore, PGE2,HGF and TGF-β1 expressions of MSC were significantly up-regulated by IFN—γ. For these three cytokines, the concentrations between experimental group and control group had statistical significance, the P value was 0.001,0.011 and less than 0.001 respectively.The MNCs obtained from peripheral blood proliferated in the presence of anti-human CD3 mAb and anti-human CD28 mAb. When the concentrations of HGF were10ng/ml,20ng/ml and 40ng/ml, the inhibitory rates of T cell proliferation were (17.4750±9.8577)%,(27.7183±8.6342)% and (36.6233±11.8959)% respectively. With the increased concentration of HGF, the inhibitory rate also increased.When HGF concentration was 40ng/ml, the inhibitory rate of T cell proliferation was significantly higher than the 10ng/ml HGF group (P=0.005).When the concentrations of TGF-β1 were 500pg/ml, 1000pg/ml and 2000pg/ml, the inhibitory rates of T cell proliferation were (13.2163±6.0858)%,(20.6529±7.3306)% and (32.5013±11.2595)% respectively. With the increased concentration of TGF-β1, the inhibitory rate also increased.When TGF-β1 concentration was 2000pg/ml, the inhibitory rate of T cell proliferation was significantly higher than the 500pg/ml group (P<0.001) and the 1000pg/ml group (P=0.015).3 T lymphocytes proliferated under the stimulation of anti-human CD3 mAb and anti-human CD28 mAb. When exogenous kynurenine (100Mm) was added into the culture system, the rate of T cell proliferation was significantly restrained (P=0.004).The expression of IDO mRNA was unable to be detected when MSC was cultured alone. In contrast, The IDO mRNA expression was remarkably enhanced when MSCs cultured in the presence of IFN-γ. Even the concentration of IFN-y was lng/ml, the IDO mRNA expression still could be detected.4 Bone marrow-derived MSC remarkably suppressed allogeneic T cell proliferation in vitro. With the increased concentration of MSC, the inhibitory rate was also increased. The suppressive role of MSC had no major histocompatibility complex (MHC) restriction.IFN-y did not break, but promoted the immunosuppressive capacity of human MSC. Addition of exogenous IFN-y (100ng/ml) into the T cell culture system had no significant effect on the inhibitory capacity of MSC (P=0.272).By contrast,5μg/ml IFN-y mAb was added into the culture system, the inhibitory rate of T cell proliferation was significantly decreased (P=0.002). Therefore, T cell proliferation was partially restored.Conclusion1 Human MSC constitutively expressed PGE2-. HGF and TGF-β1 at immunosuppressive concentrations. It was maybe one of the main mechanisms in MSC immunosuppressive activity. The pro inflammatory cytokine IFN-y did not ablate MSC inhibition of T cell proliferation but up-regulated PGE2,HGF and TGF-β1, confirming the synergistic effects of IFN-y with MSC in immunosuppression.2 IFN-y induced MSC expression of IDO, involved in tryptophan catabolism. This activity also contributed to MSC immunosuppressive effect.3 Human bone marrow-derived MSC notably suppressed allogeneic T cell proliferation in vitro. The suppressive role of MSC had no MHC restriction. Exogenous IFN-γdid not break, but promoted the immunosuppressive capacity of human MSC. Addition of IFN-y mAb into the culture system partially restored the T cell proliferation. ObjectiveTo investigate the prognosis and changes in hepatitis B serologic markers in patients whose donors or themselves were infected with HBV before allogeneic hematopoietic stem cell transplantation (allo-HSCT).MethodsWe analyzed retrospectively the clinical outcomes of 79 patients receiving allo-HSCT in our hospital between September 1992 and November 2008, including 55 patients with HBV infection and 24 patients with HBV infected donors.Results1 HBV infection did not interfere with the clinical outcomes of allo-HSCT.2 In HBsAg+group,13 (65.0%) patients developed HBV reactivation between 0.5 and 10 months after transplantation,9 (45.0%) of them developed HBV-related hepatitis.3 For the 35 HBsAg- patients with HBcAb/HBeAb positivity,4 (11.4%) of them were observed for HBV seroconversion (HBsAg became positive after immunosuppressive therapy),1 patient was concomitant with severe chronic graft-versus-host disease (cGVHD).4 There was a significant difference in HBV recativation rate between the HBsAg+and HBsAg- group (P<0.01). The incidence of hepatitis occurred within 100 days after HSCT was also higher in HBsAg+patients (P<0.05)5 2 HBsAg+patients were observed for clearance of HBV through adoptive immunity transfer, both donors were positve for HBsAb.Conclusion1 Donors or recipients infected with HBV was not considered an absolute contraindication to HSCT.2 HBsAg positivity was a high risk factor for HBV reactivation. Prophylactic lamivudine treatment could be helpful.3 For the patients with HBcAb/HBeAb positivity, HBV seroconversion could be observed, especially in patients following immunosuppressant withdrawal. Therefore, it was critical to pay close attention to the changes of HBV serologic markers in them.4 Adoptive immunity transfer was effective in clearing HBV infection in patients undergoing allo-HSCT... |
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