Studies Of Biological Significance Of PD-L1Expressed On Human Placenta-Derived Mesenchymal Stem Cells

Objective:To investigate the the biological significance of PD-L1expressed on human placenta mesenchymal stem cells (HPMSCs), including its effects on adhesion and migration of HPMSCs and its function in the immunoregulatory effects of HPMSCs on T cells.Methods:(1) Isolasion and identification of HPMSCs:Cells were isolated from healthy human placenta by the method of digestion, cultured in vitro and used after the third passage. The surface markers on the cells were identified by immuno-fluorescence and Flow cytometry (FCM). And the expression of PD-L1was analysed by FCM and RT-PCR;(2) PD-L1blockade:Specific siRNA of PD-L1were transfected into HPMSCs via liposome method. Then RT-PCR was used to detect the expression of PD-L1gene in HPMSCs.(3) Analysis of HPMSCs adhesion and migration:The adhesion rate of HPMSCs was determined by cell count, and the migration numbers of HPMSCs under the culture conditions of DMEM-LG complete medium, DMEM-LG complete medium (with SDF-la) and HPMSCs culture supernatant were evaluated using a Transwell cell culture system;(4) Isolation and coculture of PBMC:PBMC were isolated from healthy human peripheral blood by gradient centrifugation. Then PBMC, stimulated by PHA or PMA, were cocultured with PD-L1blockade HPMSCs.(5) Analysis of T cells activation phenotype, proliferation, cell cycle and IL-17secretion:Immuno-fluorescence and FCM was used to analyze the expression of early activation phenotype on T cells and the cycle of T cells. The proliferation of T cells was detected by CFSE staining. And IL-17secretion of T cells was assayed by intracellular staining.Results:(1) MSCs derived from human placenta showed plastic adherence and typical fibroblastic morphology, and expressed CD44, CD105, CD166rather than CD14, CD34and CD45.(2) PD-L1was shown to highly express on HPMSCs. Transfection of PD-L1siRNA into HPMSCs by liposome method was successful and resulted in efficient knockdown of PD-L1expression.(3) The difference of HPMSCs adhesion rate between blockade group and control group was no statistically significant after half an hour’s inoculation; but after one hour’s or three hours’ inoculation, the adhesion rate of hPMSCs was significantly higher in the blockade group than that in the control group (p<0.05). Transwell cell culture system assay indicated that HPMSCs migration numbers in blockade group were significantly reduced under the conditions of DMEM-LG complete medium, DMEM-LG complete medium (with SDF-1α) or HPMSCs culture supernatant compared with the control group (p<0.01).(4) The expression of CD69on T cells had no significantly difference in the blockade group and the control group;(5)The proliferation of T cells in blockade group, which was significantly increased compared with unblockade group, remained less than that in PMA stimulation group; The number of T cells in G0/G1phase was decreased while that of T cells in S phase was increased, but it still differed from that in PMA stimulation group; HPMSCs caused a sharp increase of IL-17secretion which was further upregulated in blockade group.Conclusion:HPMSCs highly expressed PD-L1, which could be blocked by liposome transfection of siRNA into HPMSCs. It was shown that the blockade of PD-L1could enhance the adhesion ability but reduce the migration ability of HPMSCs, and partly reverse the inhibitory effect of HPMSCs on T cell proliferation and cell cycle. In the mean while, the level of IL-17secreted by T cells was further promoted. These findings suggested that:(1) PD-L1involved in the adhesion and migration of HPMSCs;(2) PD-L1could promote the inhibitory effect of HPMSCs on T cell proliferation and cell cycle;(3) HPMSCs is able to upregulate the level of IL-17secreted by CD4+T cells, in which PD-L1played an inhibitory role;(4) PD-L1had no significant effect on the expression of CD69on actived T cells.