Effect Of Anti-CD47 Monoclonal Antibody On Acute GVHD In Haploid Transplanted Mice And Its Mechanism

ObjectiveTo investigate the effect of blockade of Integrin Associated Protein(CD47)and its ligand pathway on aGVHD model in mice with acute graft-versus-host disease(aGVHD)after haploid transplantation in order to study the role of CD47 in aGVHD In the regulation from the role.And block the pathway of Integrin Associated Protein(CD47)and its ligands in the mouse model of acute graft-versus-host disease(aGVHD)after haploid transplantation to explore the specific role of CD47 mAb in aGVHD mechanism.MethodsMale C57 BL / 6 mice were used as donor mice and female mice of hybrid C57 BL / 6 * BALB / C male C57 BL / 6 and female BALB / C mice were recipients.The hybrid generation of female mice were divided into two groups,the group A1 was given the treatment of bone marrow cells and splenocytes of the donor mice by the tail vein infusion,the group A2 was given the tail vein infusion of donor bone marrow cells and spleen cells of mice,and CD47 monoclonal antibody(B6H12),the survival time of each group,PAFS score of clinical symptoms of aGVHD(one of the indexes to evaluate the clinical symptoms of aGVHD,including posture,vitality,hair removal,dorsal bowel)Body weight curve and pathological observation of the skin,liver,small intestine and other target organ damage.After transplantation,the spleens of each group of mice were collected on + 3d days to make a single cell suspension,and the peak of CFSE of MHC-I + CD3 + cells was measured.At the same time,the spleen-derived mononuclear cells,Annexin-V and PI flow cytometry were used to detect the apoptosis of MHC-Ⅰ + CD3 + cells in each group.Spleens of recipient mice were harvested and RNA was extracted.Quantitative PCR-bet,C-maf,GATA3 expression levels,and the application of Western blotting technology verification.At the same time,the supernatant of peripheral blood of mice in each group were collected,and the secretion of cytokine(IFN-γ)by Th1 cells and the secretion of Th17-secreting cytokines(IL-17)Anti-inflammatory mechanisms in aGVHD.ResultsThe median survival of mice in group A1 was 35(24-44)days,all of whom died of severe aGVHD.The median survival time of mice in group A2 was 43(31-59)d,which was significantly different from that of group A1(P <0.01).The average body weight of mice in A1 group was(12.0 ± 0.1)g,while the average body weight of mice in A2 group was(13.6 ± 0.4)g,there was significant difference in the weight of mice in group A1(P < 0.01).The A1 and A2 mice all showed the typical clinical symptoms of a GVHD in 14 days: dorsal bowel,hair loss,apathetic,diarrhea and so on.The average PAFS score of mice in A1 group was 12.8(8-18),while that in A2 group was 8(3-13),the difference was statistically significant(P <0.01).The pathological findings of mice in group A1 were in line with the changes of a GVHD in ++ ~ +++ degrees,with vacuolar degeneration or necrosis of the basal cells of the skin,sponge layer edema and epithelial cell necrosis,separation of the focal epithelium and dermis,Between the ductal degeneration or necrosis in the 25%-75% area,small intestinal epithelial necrosis,focal mucosal nudity.The pathological findings of A2 mice were in accordance with the changes of aGVHD of + ~ + + degree.The skin of the non-focal epithelium was separated from the dermis.The area of single hepatic lobule deformity or necrosis was less than 50% and no intestinal mucosa was exposed.The results showed that the cell population of CD3 + CFSE-in group A1 and A2 were(84.83±2.32)% and(82.67±2.54)%.No significant difference between the two groups.The apoptotic state of spleen lymphocytes was detected by flow cytometry at 10 days after transplantation.The frequency of CD3 + Annexin-V + PI-cell population in groups A1 and A2 were(9.76±6.57)%、(13.77±5.21)%,The difference between the two groups was statistically significant(P <0.05).The expression of T-bet mRNA in A1 and A2 groups was(1.86 ± 0.67)and(0.39 ± 0.18),respectively,by quantitative PCR after two days(1.57 ± 0.32)and(0.95 ± 0.44),respectively,with significant difference between the two groups(P <0.05).The difference between the two groups was statistically significant(P <0.05).A1 The expression of C-maf mRNA in A2 group was(0.97 ± 0.21)and(0.30 ± 0.02),respectively.The difference between the two groups was statistically significant(P <0.05).The concentration of IFN-γ in A1 group was(52.3 ± 12.1)pg / ML after serum was detected,while the concentration of IFN-γ in A2 group was decreased to(34.3 ± 6.27)pg / ML Statistical significance(P <0.05).The concentration of IL-4 in group A1 was(199.2 ± 53.4)pg / ML,while the level of IL-4 in group A2 was(271.4 ± 59.83)pg / ML,the difference was statistically significant <0.05).The concentration of IL-17 a in group A1 was(395.3 ± 47.85)pg / ML,while the level of IL-17 a in group A2 was decreased to(256.77 ± 49.27)pg / ML,the difference was statistically significant(P < 0.05).ConclusionsInfusion of CD47 monoclonal antibody can prolong the survival time of aGVHD mice,and reduce the pathological manifestations of aGVHD and related clinical symptoms.In addition,the proliferation of donor T lymphocytes was not significantly affected during the intervention of CD47 monoclonal antibody against aGVHD,but it promoted the apoptosis of donor T lymphocytes.In addition,the expression of cytokines il-4 in Th2 was increased,and the expression of cytokines il-17 a was decreased in the Th-1 cytokine IFN-gamma and Th-17.The mRNA expression of transcription factor t-bet,GATA-3 and c-maf was also reduced.