The Subset Changes Of Dendritic Cells In Graft Versus Host Disease And Regulation Mechanism

Objective:Many studies have shown that the occurrence of graft-versus-host disease after hematopoietic stem cell transplantation is significantly related to the immune reconstitution of dendritic cells.However,the mechanism of abnormal immune reconstitution of donor dendritic cells is still unclear.The answer is that the generation and development of dendritic cells is mainly derived from hematopoietic stem progenitor cells in the bone marrow such asmulti-potent progenitor cells(MPP)and common dendritic cell progenitor cells(CDP).There are still few studies on the changes of these stem progenitor cells during the development of GVHD and their possible mechanisms.This study will explore the immune reconstruction and GVHD of dendritic cells after hematopoietic stem cell transplantation through the detection of clinical samples and a series of in vitro experiments Correlation and possible mechanisms that lead to abnormal immune reconstruction of dendritic cells.Methods:1.Use flow cytometry to detect the percentage and number of DCs and their subsets in peripheral blood of patients after hematopoietic stem cell transplantation at the time of granulocyte engraftment.2.Flow cytometry was used to detect the percentage and number of hematopoietic stem progenitor cells in bone marrow of patients 1 month after hematopoietic stem cell transplantation.3.Flow cytometry was used to detect the expression level of the functional product H3k79me2 of Dot1L in hematopoietic stem progenitor cells.4.Flow cytometry was used to detect the percentage and number of granulocytes,monocytes,and dendritic cells after 14 in vitro cultures using different cytokine combinations.5.Add Dot1L inhibitor SGC0946 to the in vitro culture system,and detect the percentage and number of granulocytes,monocytes,and dendritic cells on the 8th,12th,and 16th day of the in vitro differentiation culture of the inhibitor group and the control group.6.Sort the inhibitor group and control group with pDC and CD1c+DC after 14 days of in vitro culture using a sorting flow cytometer,co-cultivate them with CD4+T cells,and detect their secretory cells using a flow cytometer.Factors.7.Flow cytometry was used to detect the expression of co-stimulatory molecules in CD1c+DC and pDC after differentiation culture.8.Quantitative PCR was used to detect the expression of CD1c+DC and pDC secreted cytokine genes after culture differentiation.9.Quantitative PCR was used to detect the expression of DC-related transcription factors on day 4 and day 8 of in vitro culture differentiation.10.Statistical analysis Statistical analysis was performed using SPSS 22.0 statistical software.The percentage of dendritic cells and its subsets in peripheral blood,the proportion of hematopoietic stem progenitor cells in bone marrow,and the expression level of H3k79me2 in clinical samples were tested using Mann-Whitney U.A two-sided t-test was used to analyze the differences in cell numbers between groups after in vitro differentiation.And the differences in gene expression between the two groups.The difference was statistically significant using P<0.05.Results:1.Patients with 2-4 degrees aGVHD had lower percentage and number of DCs at the time of granulocyte engraftment than patients with 0-1 degrees aGVHD.The difference was statistically significant.(P<0.05)Compared with patients with 2-4 degrees aGVHD in DC subsets,patients with 0-1 degree aGVHD had lower percentage of pDC in total DCs at the time of granulocyte engraftment.The difference was statistically significant.(P<0.05)and the percentage and number of CD1c+ DC in DC were not different between the 0-1aGVHD group and the 2-4aGVHD group.(P>0.05)2.Proportion of total CD34+cells and hematopoietic progenitor cells MPP and CDP in bone marrow of patients with 2-4 degrees aGVHD at 1 month after hematopoietic stem cell transplantation compared with patients with 0-1 degree aGVHD And the number is small,the difference is statistically significant.(P<0.05)3.In patients with 2-4 degrees aGVHD,the expression of H3k79me2 in MPP and CDP in bone marrow was lower than in patients with 0-1 degrees aGVHD.The difference was statistically significant.(P<0.05)4.The combination of MS-5+GFS3 can more effectively differentiate hematopoietic stem cells into DC in vitro.5.Dot1L inhibitor SGC0946 can reduce the proliferation of hematopoietic stem cells in vitro,at the same time can promote the differentiation of hematopoietic stem cells to granulocytes,and inhibit the differentiation of hematopoietic stem cells to pDC.The difference is statistically significant.(P<0.05)6.Compared with the control group,the DCs obtained by using Dot1L inhibitor SGC0946 can promote the differentiation of CD4+T cells into Th1 and Th2.7.Differentiated DCs cultured with Dot1L inhibitor SGC0946 have higher expression levels of the co-stimulatory molecules HLA-DR,CD86,and CD83 than the control group.8.Compared with the control group,the DCs differentiated from the Dot1L inhibitor SGC0946 had lower cytokine IL-12,IFN-?,and IFN-? gene expression than the control group.9.The expression of IRF4,BATF3,and FLT3 genes on the fourth day of culture with Dot1L inhibitor SGC0946 was significantly lower than that of the control group,and the difference was statistically significant.(P<0.05)TCF4,IRF4,IRF8,and FLT3 gene expression levels were significantly lower than those of the control group on the eighth day,and the difference was statistically significant.(P<0.05)Conclusion:1.Patients with GVHD have impaired DC reconstruction,especially pDC,in peripheral blood when granulocytes are engrafted.2.The decrease of MPP/CDP in hematopoietic stem progenitor cells in bone marrow of patients with GVHD is significantly related to the occurrence of 2-4 severe GVHD reactions.This phenomenon is related to the low expression of Dot1L in hematopoietic stem progenitor cells.3.Compared with other cytokines,MS5+GSF3 in vitro differentiation culture system could better differentiate hematopoietic stem cells to DC.4.Inhibition of the methylation function of Dot1L in hematopoietic stem cells will lead to a decrease in the expression of transcription factors related to DC differentiation,which will affect the number of DCs and especially pDCs.It will also be beneficial to DC-induced CD+T activation and co-stimulation molecular expression.