The Study Of The Mechanism Of IL-10~+T Cell Differentiation Induced By Human Placenta-derived Mesenchymal Stem Cells And Its Application In Treating EAE

1. The role of PDL1 in the differentiation of IL-10+T cells from cord and peripheral blood-derived T cells induced by hPMSCs.Objective: To compare the differentiation, inducing effect of human placenta derived mesenchymal stem cells (hPMSCs) on IL-10+T cells derived from cord blood and peripheral blood, and investigate the mechanism in the induction.Methods: (1) The hPMSCs were isolated via enzyme digestion and then cultured.After the third passage, the morphology of cells and the surface markers on it were detected. (2) The expression of PDL1 in hPMSCs were detected by RT-PCR and FCM respectively. (3) Mononuclear cells were isolated from cord blood and peripheral blood from healthy donors by Ficoll density gradient centrifugation and T cells were purified by sheep red blood cells. (4) Then hPMSCs, pre-treated with or without Anti-PDL1mAb, were co-cultured with PHA activated T cells. Percentages of CD4+IL-10+T cells and CD8+IL-10+T cells in cord blood and peripheral blood T cells were analyzed by FCM.Results: (1) The ratios of CD4+IL-10+T cells and CD8+IL-10+T cells in cord blood and peripheral blood T cells activated by PHA were significantly increased in the presence of hPMSCs compared with the absence of hPMSCs groups (P<0.01). (2)FCM results showed that the ratios of CD4+IL-10+T cells and CD8+IL-10+T cells from hPMSCs co-cultured with activated peripheral blood T cells group were higher than that from hPMSCs co-cultured with activated cord blood T cells group. (3) PDL1 was highly expressed in hPMSCs. While after blocking its expression in hPMSCs,the percents of CD4+IL-10+T cells and CD8+IL-10+T cells were significantly reduced in cord blood and peripheral blood T cells.Conclusion: PDL1 was involved in the differentiation of IL-10+T cells from cord blood and peripheral blood T cells induced by hPMSCs. Meanwhile the ability of hPMSCs to induce the differentiation of IL-10+T cells from peripheral blood T cells was apparently stronger than that in cord blood T cells.2. The effects of IFN-y and IL-1? on the differentiation of IL-10+T cells induced by hPMSCs.Objective: To detect the impact of IFN-? and IL-1? on the expression of PDL1 in hPMSCs, as well as their effect on the differentiation of IL-10+T cells induced by hPMSCs.Methods: (1) Anti-PDL1mAb was used to block the protein expression in hPMSCs, then the effect of PDL1 on the differentiation of CD4+IL-10+T cells and CD8+IL-10+T cells from CD3/CD28mAb activated T cells induced by hPMSCs were detected. (2) hPMSCs were pre-stimulated with IFN-? and IL-1?, then the levels of PDL1 were detected by qRT-PCR and FCM, respectively. (3) FCM was used to detect the percentages of IL-10+T cells in activated T cells co-cultured with hPMSCs with or without IFN-? and IL-1? pre-treated.Results: (1) The percents of CD4+IL-10+T cells and CD8+IL-10+T cells were obviously decreased in CD3/CD28mAb activated T cells with hPMSCs and Anti-PDL1mAb than that without blocking mAb. (2) After pre-treated with IFN-? and IL-1? alone or in combination, the expression of PDL1 in hPMSCs, as well as the ability of hPMSCs in inducing the differentiation of IL-10+T cells were significantly higher than that of no stimulation, and jointly pre-treated group was higher than lonely pre-treated group.Conclusion: IFN-?, synergy with IL-1? could up-regulate the expression of PDL1 in hPMSCs, thus promoting the differentiation of IL-10+T cells induced by hPMSCs.3. hPMSCs could alleviate the symptoms of EAE by increasing the percents of IL-10+T cellsObjective: To observe whether transplanted hPMSCs could alleviate the symptoms of experimental autoimmune encephalomyelitis (EAE) which is the classical model of multiple sclerosis (MS), as well as explore the mechanism in it.Methods: (1) EAE model was induced in female C57BL/6 mice with immunization of myelin oligodendroglia glycoprotein (MOG) through subcutaneous injection (first injection day is day 0). Then behavior score was evaluated daily until 25 days. (2) Phosphate buffered saline (PBS) and hPMSCs were injected via tail vein at day 14. (3) Hematoxylin and eosin (HE) and luxol fast blue(LFB) staining were used to evaluate the presence of lymphocyte infiltration, as well as demyelination changes, in spinal cord before and after the treatment of transplanting hPMSCs. (4) The percent of IL-10+T cells in the spleen cells were detected by FCM at day 14 and day 25.Results: (1) Mice treated with hPMSCs showed a lower behavior scores compared with that treated with PBS (P<0.05). (2) HE results showed that in hPMSCs-treated mice the lymphocyte infiltration and demyelination were significantly reduced than that in EAE mice (P<0.05). (3) Results from FCM assays showed that the percent of IL-10+T cells in the spleen of EAE-treated mice at day 14 were significantly decreased than that in control mice. Meanwhile, these percents present a little increase at day 25. However, after transplanting hPMSCs these percents were significantly increased as compared with that in mice receiving PBS(P<0.05).Conclusion: hPMSCs could increase the percents of IL-10+T cells in EAE mice and then improve its symptoms.