Immunomodulatory Effect Of Mesenchymal Stem Cells On CD4~+T Cells In Type 1 Diabetic Mice

BackgroundT1D is a chronic metabolic disease caused by autoimmune system.The main pathogenesis of T1 D is the over-response of the immune system leading to the destruction of pancreatic ? cells.CD4+T cells,especially Th1 and Tregs cells,play an important role in destroying pancreatic ? cells.MSCs can immunoregulatory T cells.MSCs can inhibit T cells by secreting soluble factors and direct contact between cells.As a membrane protein,PD-L1 plays a key role in inducing T-cell anergy and maintaining peripheral immune tolerance.Studies have shown that the PD-L1/PD-1 pathway plays an important role in the immunosuppression of immune cells by MSCs.The way in which MSCs can regulate T1 D CD4+T cells and the molecular mechanism still need to be studied.In this study,we first explored the difference between CD4+T cells and their subtypes Th1 and Tregs cells in normal and T1 D spleen cells by flow cytometry.Next,we investigated the effect of co-culture of BMSCs with T1 D spleen cells on the proportion of CD4+ T cells and their subtype Th1 and Tregs cells in spleen cells.We also explored the expression of PD-L1 of BMSCs and PD-1 of spleen cells after co culture of BMSCs and T1 D spleen cells.Finally,the PD-L1 blocking antibody was added to the BMSCs and T1 D spleen cells co culture system to explore the role of PD-L1/PD-1 pathway in the regulation of BMSCs on the proportion of Th1 and Tregs cells in T1 D spleen cells.ObjectiveBMSCs were co-cultured with T1 D spleen cells to investigate the immuneregulation role and mechanism of BMSCs on T1 D CD4+ T cell.Methods1.Male C57BL/6J mice were used to establish a T1 D mouse model by intraperitoneal injection of streptozotocin,and the mice's fasting blood glucose and body weight were measured.The pancreas tissues of the normal and the T1 D mice was extracted.Hematoxylin eosin staining of mice pancreas was used to observe the difference of pancreatic tissues structure between the normal and the T1 D mice.The normal and T1 D spleen cells were extracted by tissue grinding method.The spleen cells were labeled with CD4-PE.The difference between the proportion of CD4+T cells and their subtypes Th1 and Tregs cells in normal and T1 D spleen cells was detected by flow cytometry.2.Extraction of normal mouse BMSCs by whole bone marrow adherent culture.BMSCs and T1 D spleen cells were co cultured to explore the immunoregulatory effect of BMSCs on spleen cells.Since BMSCs can immunomodulate T cells by secreting soluble factors(indirect contact)and direct contact with T cells.BMSCs and spleen cells were co-cultured with transwell chambers to investigate the immunomodulatory effects of BMSCs and spleen cells under indirect co-culture conditions;BMSCs were co-cultured with spleen cells to investigate the immunomodulatory effects of BMSCs and spleen cells in direct contact.The groups were divided into a.Spleen cells(normal),b.Spleen cells(T1D),c.Spleen cells(T1D)+ BMSCs,d.Spleen cells(T1D)+ BMSCs(transwell chambers).The spleen cells were labeled with CD4-PE to detect the effect of BMSCs co culture on the proportion of CD4+T cells in spleen cells.The spleen cells were labeled with CD4-PE,IFN-?-APC and CD4-FITC,CD25-PE-Cy7,Foxp3-PE,and the changes in the proportion of Th1 cells and Tregs cells in the T1 D spleen cells were detected after co culture with BMSCs.Extraction of RNA and protein from BMSCs and spleen cells in co-culture system,and the changes of PD-L1 of BMSCs and PD-1 of spleen cells at m RNA and protein levels were detected by RT-q PCR and Western-Blot after BMSCs and T1 D spleen cells co culture.3.PD-L1 blocking antibody was added to the co-culture system of BMSCs and T1 D spleen cells,and the ratio of Th1 and Tregs cells was detected by flow cytometry.Result1.Compared with normal mice,the islet tissue of T1 D mice was destroyed,and the proportion of CD4+T cells in the T1 D spleen cells increased and the proportion of subtype Th1 cells increased(P<0.001),and the proportion of Tregs cells decreased(P<0.001).2.When the T1 D spleen cells co-cultured with BMSCs directly,the proportion of Th1 decreased(P<0.001)and the proportion of Tregs increased.However,no significant changes occurred in the transwell chamber.At the same time,the expression of PD-L1 of BMSCs and PD-1 of spleen cells in the co-culture system of T1 D spleen cells and BMSCs was increased at the RNA and protein level(P<0.001).3.When PD-L1 blocking antibody was added to block the PD-L1/PD-1 pathway in the direct co-culture system of BMSCs and T1 D spleen cells,the proportion of Th1 and Tregs in the co-culture system did not change compared with T1 D spleen cells.Conclusion1.Compared with normal spleen cells,T1 D spleen cells have a high proportion of CD4+ T and Th1 cells and a low proportion of Tregs.2.BMSCs down-regulate the ratio of Th1 cells and upregulate the proportion of Tregs by direct contact with T1 D spleen cells.3.The PD-L1/PD-1 pathway plays an important role in BMSCs down regulation of T1 D spleen Th1 cells and upregulation of Tregs cells.