Exploration Of The Mechanism Of NK Cells And Macrophages In Chronic Allograft Rejection

Chronic rejection is the main cause of organ failure in long-term transplant patients.In earlier studies,adaptive immune cells were considered to be the main cell type involved in chronic rejection.With the emergence of immunosuppressive agents,adaptive immune responses in the allo-transplantation have been controlled,which has greatly improved the survival rate of patients after transplantation,but the long-term survival rate of transplanted organ is still low.Thus,the roles of innate immune cells in chronic rejection have drawn more attention.In recent years,the results of animal experiments and clinical data have shown that innate immune cells,especially natural killer cells(NK cells)and macrophages,play an important role in chronic transplant rejection.However,when T and B cells are deficient,whether NK cells and macrophages play an independent role in chronic rejection of transplantation or reduce the occurrence of chronic rejection through mutual regulation,the mechanism remains to be determined.Objective:To explore the establishment of chronic rejection model by skin parental-to-offspring transplantation,by analyzing the proportion of NK cells and macrophages in spleen and lymph nodes with flow cytometry(FCM)at different time points after skin transplantation and the infiltration of NK cells and macrophages in skin grafts through immunohistochemistry,to understand how the NK cells and macrophages change and to get to know the role of NK cells and macrophages in chronic rejection.In addition,to investigate the mechanism of NK cells and macrophages in chronic rejection through allogenic heart transplantation model with severe combined immunodeficiency(SCID)mice in combination with in vitro experiments.Methods:1.Changes of NK cells and macrophages in mice at different time after transplantation.The tail skins of 6-8 weeks old C57BL/6 mice or F1(C57BL/6×BALB/c)mice were transplanted to the back of F1 mice.Spleen,lymph nodes,peripheral blood and the skin grafts were taken from recipient mice at different time points(D1,D2,D3,D5,D7,D10,D14 and when the skin graft were rejected)after transplantation.The proportion of CD4~+T cells,CD8~+T cells,NK cells and macrophages in spleen,lymph nodes and peripheral blood were detected by FCM.The infiltration of lymphocytes in skin graft at different time points was detected by Hematoxylin-eosin(HE)staining.The infiltration of NK cells and macrophages in skin graft was detected by immunohistochemistry.2.Exploration of the mechanism of NK cells and macrophages in a mouse heart transplant model.The grafts were removed from 6-8 weeks old C57BL/6mice and stored at 4°C for 4 hour before transplantation,and then were transplant into the 6-8 weeks old NOD/SCID and CB17/SCID mice.Spleen and cardiac grafts were taken from recipient mice at day 60 after transplantation.Splenocytes from recipient mice were analyzed by FCM for T cells,B cells,NK cells and macrophages and the subsets of NK cells and macrophages.The pathological character of cardiac grafts were observed with HE staining.The expression of NK cells,macrophages and IL-6 in cardiac grafts tissue were detected by immunohistochemistry.In vitro experiments,the expression of the NK cell activating receptors(CD69,NKp46,NKG2D)and inhibitory receptor Ly49A were detected by FCM,and the level of IL-6 and IFN-γin supernatants was detected by ELISA.3.As it was found that NOD/SCID mice and CB17/SCID mice used in the experiment unexpectedly showed very similar rejection,we comparative study on the activity of NK cells in NOD/SCID and CB17/SCID mice.Splenocytes from naive mice were stained with fluorescent conjugated antibody targeting CD3,CD19,NK1.1 and DX5 for phenotyping.Splenocytes were isolated from poly(I:C)treated mice were stimulated with cytokines(IL-2,IL-12,IL-15)or naive mice for analysis of the functional molecules of NK cells.Splenocytes and purified NK cells from poly(I:C)treated mice were stimulated with cytokines(IL-2,IL-12,IL-15)and then mixed with CFSE-labeled YAC-1 cells for cytotoxicity assay in vitro.Splenocytes from naive and poly(I:C)treated mice,respectively,were stimulated with cytokines(IL-2,IL-12,IL-15)in vitro for IFN-γproduction assay by FCM or ELISA.Results:1.Changes of NK cells and macrophages in mice at different time points after transplantation.The percentages of CD4~+T cells,CD8~+T cells,NK cells and macrophages in spleen and lymph nodes from transplanted mice at different time points after skin transplantation were detected by FCM.The results showed that:In both the spleen and lymph nodes,allogeneic transplantation group(C57BL/6 to F1)compared with syngenic transplantation group(F1 to F1),the percentage of CD4~+T cells,CD8~+T cells,NK cells and macrophages at different time points after skin transplantation,there was no significant difference in the regularity,suggesting that parent-to-offspring skin transplantation could not cause the regular changes of immune cells in the spleen and lymph nodes at the early stage after transplantation.Three mice developed chronic rejection during 30-40 days after skin transplantation,the results of FCM analysis of the proportions of CD4~+T cells,CD8~+T cells,NK cells and macrophages in spleen,lymph nodes and peripheral blood showed that there was no significant difference between the mice with and without chronic rejection.The result of HE staining of skin graft showed that there was a large amount of lymphocyte infiltration in the skin graft in both syngeneic transplant group and allogeniec transplant group at day 1-5 after transplantation.However,from day 7 to 14,there was larger amount of lymphocyte infiltration in the skin graft in allogeneic transplant group,compared with the syngeneic transplant group,but this phenomenon only existed in a few transplanted mice.Immunohistochemical detection showed that NK cells and macrophages were infiltrated in the rejected skin graft,suggesting that both NK cells and macrophages were involved in chronic rejection.2.Exploration of the mechanism of NK cells and macrophages in a mouse heart transplant model in mice.The FCM results showed that NOD/SCID and CB17/SCID mice after heart abdominal heterotopic transplantation were T cells and B cells deficient,without"leaky".Compared with the syngeneic transplant group(CB17/SCID to CB17/SCID),thickened artery wall in cardiac grafts from recipient mice can be observed after the H&E stained from both allotransplant groups(C57BL/6 to CB17/SCID and C57BL/6 to NOD/SCID)at D60 after transplantation.The FCM results showed no significant difference of NK cells,macrophages and their subsets among the three transplant groups(the two allogeneic transplant groups and the syngeneic transplant group)at D60.Immunohistochemical results showed that there were a larger number of NK cells and macrophages and increased expression of IL-6 in the cardiac grafts fom allogeneic transplant mice compared with that from syngeneic transplant mice,suggesting that NK cells,macrophages and IL-6 may be involved in the occurrence of chronic rejection.The results of the experiment in vitro showed that compared with the NK cell culture group,the expression of CD69 of NK cells in the mixed culture group(C57BL/6 mouse NK cells and BALB/c mouse macrophages)was significantly increased,the expression of NKp46,NKG2D and Ly49A were no difference between the two groups.In the supernatant,the level of IL-6 or IFN-γin the mixed culture group was significantly higher than the sum of the level of corresponding cytokine in NK cells culture alone group and macrophages culture alone group.3.Comparative study on the activity of NK cells in NOD/SCID and CB17/SCID mice.The proportion of NK1.1 and DX5 in splenocytes of C57BL/6,BALB/c,NOD/SCID and CB17/SCID mice were detected by FCM,and the results showed that there was no NK1.1 positive cell population in splenocytes of the other three strains except C57BL/6 mice.The proportion of splenic NK cells did not show significant difference between in naive NOD/SCID and CB17/SCID mice.The CD69,Fas L,TRAIL,granzyme B,perforin and NKp46 levels in splenic NK cells were similar between the naive three strains mice,while the NKG2D levels in NK cells from NOD/SCID and BALB/c mice were higher than that from CB17/SCID mice.Moreover,the NKG2A/C/E levels in NK cells from naive NOD/SCID were significantly lower than that from CB17/SCID and BALB/c mice.The Ly49A levels in NK cells from NOD/SCID mice were higher than that from CB17/SCID and BALB/c mice.In the cytotoxicity assay,the splenocytes or purified NK cells were stimulated with cytokines(IL-2,IL-12,IL-15)of the poly(I:C)-treated CB17/SCID and NOD/SCID mice,the splenocytes from CB17/SCID mice showed higher cytotoxicity than that from NOD/SCID mice.However,the cytotoxicity of purified NK cells from NOD/SCID mice was similar to that from CB17/SCID at most of effector-to-target ratios except for the lowest one.Under the same conditions as the cytotoxicity assay,the levels of perforin and NKp46 were similar in NK cells between the CB17/SCID and NOD/SCID mice,while the granzyme B and NKG2A/C/E levels in NK cells from NOD/SCID mice were significantly lower than those from CB17/SCID mice.Moreover,the NKG2D and Ly49A levels in NK cells from NOD/SCID mice were higher than those from CB17/SCID mice.After in vitro stimulation with cytokines,the splenocytes from CB17/SCID mice showed higher percentage of NK cells and IFN-γlevel than that from NOD/SCID mice.However,the IFN-γproduction of NK cells showed no significant difference between the two mice strains.Conclusion:1.NK cells in allogeneic skin grafts from parent to offspring are usually insufficient to be activated by the existence of"missing self"and lead to chronic rejection.NK cells and macrophages are involved in the chronic rejection.2.When T and B cells were absent,the percentage of NK cells and macrophages in spleen,lymph nodes and peripheral blood of allograft transplant recipients was not enough to reflect the possible activation differences during chronic rejection.NK cells and macrophages are involved in chronic rejection,and the two play a synergistic role,and IL-6 may be one of the important mediators of the synergistic promotion of chronic rejection.3.Compared with CB17/SCID and BALB/c mice,the function of NK cells in NOD/SCID mice was only partially compromised,so NOD/SCID mice were not suitable to be selected as NK cell-deficiency models.