The Role And Mechanism Of ??T17 Cells In Intestinal Acute Gralt-versus-host Disease

Acute graft versus host disease(aGVHD)is still one of the most serious and challenging complications after allogeneic hematopoietic stem cell transplantation(allo-HSCT).The incidence of aGVHD after allo-HSCT is about 40-60%,mainly involving the skin,gastrointestinal tract and liver.Among them,gut aGVHD is currently at high risk and still difficult to cure,and it is one of the important causes of death after allo-HSCT.However,due to factors such as difficulty in obtaining materials and less attention paid on regional research,the gut's regional environment of immunology during the development of aGVHD,the dynamic changes and interactions of immune cells remain unclear.In this study,we established a mature and stable murine allogeneic bone marrow transplantation and aGVHD model,using the intestinal mucosal single cell separation technique to detect T cells,B cells,NK cells,macrophage,myeloid-derived suppressor cell(MDSC),dendritic cell(DC),innate lymphoid cells(ILC)in the intestinal mucosa of aGVHD mice after transplantation,and focused on the variation of them and their relationship with aGVHD;Then we analyzed the changes of the important cytokines IL-17A,IL-22 and their source cells in the intestinal mucosal barrier,and found that IL-17-secreting y8T(??T17)cells are the main source of IL-17A and IL-22 in intestinal mucosa lamina propria.We identified the biological characteristics of intestinal mucosal ??T1 7 cells in bone marrow-transplanted mice by single-cell qPCR technique,and screened out the possible mechanism of ??T17 cells in the intestinal tract as well as their immunoregulatory function.Finally,this study established in vitro induction of IL-22+??T17 cell culture system,found that intestinal y8T17 cells are the key subgroup of cells in intestinal protection in aGVHD,and its regulation and related mechanisms are discussed in depth.This study has important theoretical and practical significance for revealing the immune characteristics of intestinal regions in aGVHD,discovering new strategies for potential prevention and treating new targets.Chapter 1 Study on the subpopulation of intestinal cells in mice after bone marrow transplantationBackground&Aims:At present,there are no comprehensive reports on the composition,source and changes of various immune cells in the lamina propria of intestinal mucosa during the development of aGVHD.In this part,we studied the sub-population of lamina propria cells in the intestinal mucosa of mice after bone marrow transplantation,and explored the sources and changes of common immune cells such as T cells,B cells,NK cells,MDSCs,macrophages and DCs in none-aGVHD and aGVHD mouse.Methods:We used a Balb/c background mice as a recipient to establish a stable mouse bone marrow transplantation aGVHD model with C57/B6 background mice as donors.In the second week after transplantation,mouse were randomly selected from none-aGVHD group and aGVHD group,and their intestinal tracts were observed for damage.The intestinal mucosa was separated into single cells by a mixture of collagenase,EDTA,and DNase,and the cells were filtered with different concentrations of percoll.Flow cytometry was then used to detect biological characteristics such as cell type,quantity and source.Results:After establishing a stable murine bone marrow transplantation aGVHD model,we observed aGVHD symptoms such as decreased body weight,arched back,diarrhea,and hair loss in the head,face and back.After dissection,it was found that the intestinal lumen of the small intestine became thinner,the intestinal wall was red and thin,and many dark and watery stools remained in the intestinal lumen.The intestinal mucosa lamina propria CD3+T cells,CD 19+B cells,NK cells,MDSC,macrophages,and DC mostly expressed the donor mouse C57/B6-specific protein H2kb.It was further found that the number of B cells in the intestinal mucosa of none-aGVHD mice was the most,followed by NK cells,MDSCs,DCs,T cells,and macrophages;while in aGVHD mice the T cells ranked top quantatively,followed by NK cells and MDSCs,macrophages and B cells.The same cell was compared and analyzed.The aGVHD mice had more T cells than the none-aGVHD mice,while the B cells,NK cells,MDSCs and DCs were less than the aGVHD-free mice.There was no significant difference in macrophages between the two groups.Conclusions:The majority of immune cells in the intestinal mucosa of mice after bone marrow transplantation are donor sources.When aGVHD occurs,T cells are obviously expanded,and the number of MDSCs with immunosuppressive function is obviously impaired.Chapter 2 Changes of IL-17 and IL-22 and their secretory cells in the intestinal tract of mice after bone marrow transplantationBackground&Aims:Cytokines play an important role in the development of aGVHD,and IL-17 and IL-22 are important cytokines with complex effects.At present,the secretion of IL-17 and IL-22 in intestinal mucosa in murine bone marrow transplantation model is still unclear.Therefore,this chapter will study the related biological characteristics and their quantitive changes of intestinal IL-17 and IL-22 secreting cells.Methods:After establishing a mouse allogeneic bone marrow transplantation model,mice were randomly selected from none-aGVHD group and aGVHD group in the second week after transplantation,and the intestinal mucosa lamina propria cells were taken for multicolor flow cytometry detection.The cell types and proportions of IL-17 and IL-22 secreting cells were counted.Results:There was no significant difference in the secretion of IL-17 in the lamina propria of mouse intestinal mucosa after bone marrow transplantation.The IL-22 secretion ability of mice in aGVHD group was lower than that in none-aGVHD group.In the two groups,IL-17A and IL-22 were mainly derived from donor ??T cells.In the aGVHD group,the proportion of ??T17 cells in the intestine was significantly lower than that in the aGVHD group,and the IL-22+??T17 cells in the aGVHD group were also lower than that in the none-aGVHD group.Conclusions:??T cells are the main secretory source of IL-17 and IL-22 in intestinal mucosa after bone marrow transplantation.y8T17 cells secreting IL-22 may be a key subpopulation of protective cells in intestinal aGVHD,but the specific mechanism and mechanism are still to be further studied.Chapter 3:Biological characteristics and mechanism of ??T17 cells in intestinal aGVHDBackground&Aims:??T cells are a class of non-MHC-restricted innate immune cells that are widely distributed in mucosal tissues such as the intestinal tract and the skin.They are called bridges between adaptive immunity and innate immunity.Different subpopulations of y8T cells may play different roles in pathogenesis or protection during the pathological process of aGVHD.However,the biological characteristics of intestinal y8T17 cells in mouse aGVHD and its mechanism of action with other immune cells and stromal cells,such as MDSC and IEC,have not been reported.Methods:The intestine of none-aGVHD group and aGVHD group mice were taken after transplantation,and the intestines were stained with immunofluorescence and photographed.We further seperated intestinal single cells from wild-type donor C57/B6 mice,none-aGVHD mice,and aGVHD mice.After staining,we sorted CD45+CD3+y8TCR+cells,and used single-cell qPCR to screen its phenotype and function.The spleen cells were induced by IL-1?+IL-23 combined with retinoic acid regimen to detect the secretion of IL-17 and IL-22 in ??T cells,and the aGVHD model was established by the optimal induction protocol.Results:Intestinal immunofluorescence suggested that ??TCR and IL-17A colocalized in the lamina propria of the intestinal mucosa.The results of single cell qPCR showed that ??T17 cells in mice had high expression of il17a,ill7f,il8,csf2,IL23R,Il17ra,Il17rc,Ocln as well as other genes.IL-22+ ??T17 cells were detected higher expression of Il-22r,Id2,Steap4.Tlr1,ccr9 than IL-22" y8T17 cells.When aGVHD occurred,the expression of notch2,csf2 and il22 genes in ??T17 cells were lower than those without aGVHD.The induction efficiency of IL-22+??T17 cells with 10ng/ml IL-1 and 10ng/ml IL-23 and 1?m RA culture system was the highest.Elevated IL-22+??T17 cells can reduce the severity of aGVHD and reduce the mortality of aGVHD.Conclusions:y8T17 cells exist in the lamina propria of mouse intestinal mucosa after bone marrow transplantation.IL-22+??T17 cells may be a key subpopulation of protective cells in the intestine,which may promote intestinal epithelial repair,support the tight junction in the intestinal tract,recruiting MDSC to inhibit the immune response by secreting cytokines.Il23r,Id2,Steap4,77r1,ccr9 and notch2 may be the regulatory factors involved in the regulation of IL-22 expression in y8T17 cells.IL-22+y8T17 cells have important protective effects during aGVHD.