The Mechanism Of Human Placenta-derived Mesenchymal Stem Cells Inducing The Differentiation Of IL-10~+T Cell Subsets

1. The induction effect of hPMSCs on peripheral blood T cells to IL-10~+T cell subsets.Objective:To investigate the effect of PDL2 in the induction of human placenta derived mesenchymal stem cells (hPMSCs) on peripheral blood T cells to IL-10~+T cell subsets.Methods:(1) hPMSCs were isolated from healthy full-term human placenta tissues by the method of enzyme digestion. The cells were used after the third passage, the morphology of cells was observed under the inverted phase contrast microscope and the surface markers on the hPMSCs were detected by FCM. (2) The expression of PDL2 on hPMSCs were detected by RT-PCR and FCM respectively. (3) PBMC were isolated from healthy volunteers peripheral blood by density gradient centrifugation and CD3+T cells were purified with CD3 beads. (4) Non-activated T cells were co-cultured with hPMSCs at the ratio of 10:1, and analyzed the ratio of CD4+IL-10~+and CD8+IL-10~+T cell subsets at day 1, day 3 and day 5 respectively. (5) FCM was used to detect the ratio of CD4+IL-10~+ and CD8+IL-10~+T cell subsets of PHA(72h) or CD3/CD28mAb(96h) activated T cells in the presence of hPMSCs. (6) Transwell method was uesd to detect the ratio of CD4+IL-10~+ and CD8+IL-10~+T cell subsets. (7) Using PDL2 mAb to block PDL2 protein expression on hPMSCs, and detected the effect of PDL2 in the induction of hPMSCs on peripheral blood T cells to CD4+IL-10~+ and CD8+IL-10~+T cell subsets. (8) The levels of IL-10 of co-cultured supernatant were detected by enzyme-linked immunosorbent assay (ELISA) before and after blocking PDL2 expression.Results:(1) hPMSCs showed the characteristic of plastic adherence and fibroblastic morphology; Its surface markers include CD29+?CD44+?CD105+? CD166+?CD34-?CD45- and CD14-. (2) hPMSCs highly expressed PDL2 molecules at the level of gene and protein. (3) The purity of CD3+ T cells were higher and could be used in the experiment. (4) The FCM results showed that hPMSCs could induce CD4+IL-10~+ and CD8+ IL-10~+T cell subsets differentiation, and the ratio of CD4+IL-10~+ and CD8+ IL-10~+T cell subsets of the co-culture group was significantly higher than the T cells group, and the positive rate of 5d was the highest group. (5) The ratio of CD8+ IL-10~+ T cell population in T cells activated by different stimulates, including PHA and CD3/CD28mAb were significantly increased in the presence of hPMSCs compared with the absence of hPMSCs groups (p<0.01), in addition, the ratio of CD3/CD28mAb activated group was higher than PHA activated group. (6) The results of FCM showed that the the ratio of CD4+IL-10~+ and CD8+IL-10~+T cell subsets from the transwell culture system was lower than that of direct contact system. (7) The blocking experiments indicated that the addition of the PDL2 mAb down-regulated the percent of CD4+IL-10~+ and CD8+IL-10~+T cell subsets in PHA or CD3/CD28mAb stimulated T cells in the presence of hPMSCs compared with the unblocked groups. (8) The ELISA results showed that the content of IL-10 of activated group were higher than the non-activated group in the supernatant of co-cultured system. The content of IL-10 decreased significantly after blocking PDL2 expression.Conclusion:(1) hPMSCs highly expressed PDL2. (2) hPMSCs could respectively induce activated and non-activated T cells to CD4+IL-10~+ and CD8+IL-10~+ T cell subsets, and the induction of activated group was more obvious, especially in the CD3/CD28mAb activated group. (3) hPMSCs promoted the secretion of IL-10, and PDL2 played an important role in it.2. The effects of IFN-? and TNF-? in the induction of hPMSCs on peripheral blood T cells to IL-10~+ T cell subsets.Objective:Examinations were conducted to investigate the impact of IFN-? and TNF-? on the expression of PDL2 in hPMSCs and in the induction of hPMSCs on peripheral blood T cells to IL-10~+T cell subsets.Methods:(1) The expression of PDL2 on hPMSCs with stimulation of IFN-? and TNF-? were detected by qRT-PCR and FCM respectively. (2) FCM was used to detect the ratio of CD4+IL-10~+ and CD8+IL-10~+T cell subsets with hPMSCs stimulated by IFN-? and TNF-?. (3) The levels of IL-10 of co-cultured supernatant were detected by ELISA with hPMSCs stimulated by IFN-? and TNF-?..Results:(1) After IFN-? and TNF-? stimulated hPMSCs alone and in combination, the expression of PDL2 in gene and protein levels elevated significantly, and jointly stimulated group was higher than the stimulus alone. (2) After IFN-? and TNF-? stimulated hPMSCs alone and in combination, the ratio of CD4+IL-10~+ and CD8+IL-10~+T cell subsets were significantly higher than that of no stimulation, and jointly stimulate group was higher than the stimulus alone. (3) After IFN-? and TNF-? stimulated hPMSCs alone and in combination, the content of IL-10 in the supernatant of co-cultured system increased significantly, and jointly stimulate group was higher than the stimulus alone.Conclusion:(1) IFN-? and TNF-? could up-regulate the expression of PDL2 in hPMSCs, and have a synergy between each other. (2) IFN-? and TNF-? could regulate the induction of IL-10~+T subsets through adjusting the expression of PDL2 in hPMSCs. (3) IFN-? and TNF-? might play an important role in hPMSCs promoting the secretion of IL-10.