The Effect Of TIRC7 On Acute Graft-versus-host Disease

Objective: To explore the relevance of TIRC7 with acute graft-versus-host disease(GVHD) and the pathogenesis of TIRC7 in the development and progression of acute GVHD.Methods:(1) Plasma and mononuclear cells were obtained from the peripheral blood of acute GVHD patients before and after treatment. ELISA and q-PCR were respectively used to detect TIRC7 and CTLA-4 expression level in the peripheral blood from acute GVHD patients before and after treatment in order to asertain whether TIRC7 and CTLA-4 were correlated with acute GVHD.(2) We chose the lymphocytes in patients with acute GVHD as target cells, used the methods of plasmid construction and electroporation and monitored the regulation of TIRC7 on CTLA-4 and the effect of TIRC7 on T-cell activation and the secretion of cytokines by flow cytometry and western blot and other approaches of detection.(3) SPF C57BL/6 mice were chosen as donor mice and BALB/c as recipients. In the mouse model of acute GVHD which is well established in our laboratory, we utilized TIRC7 and CTLA-4 monoclonal antibody by intraperitoneal injection. In the mouse model of acute GVHD, we observed the effect of TIRC7 and CTLA-4 on the activation of T cell and acute GVHD, respectively.Results:(1) Both TIRC7 and CTLA-4 expression were detected in acute GVHD patients by RT-PCR and western blot. The expression of TIRC7 was elevated in patients with acute GVHD as measured by ELISA and q-PCR; nevertheless, TIRC7 level was decreased on the 28 day after treatment and its expression was positively correlated with the degree of acute GVHD. CTLA-4 expression was declined in acute GVHD patients and its level was increased on the 28 day after treatment and itsexpression was negatively correlated with the degree of acute GVHD.(2) CTLA-4 expression was decreased after TIRC7 expression was down-regulated and the luciferase activity of STAT3 luciferase reporter gene plasmid was significantly decreased and meanwhile, the degree of phosphorylation of STAT3 was reduced. But the expression of TIRC7 did not change after CTLA-4 expression was up-regulated and the luciferase activity of STAT3 luciferase reporter gene plasmid was significantly increased and meanwhile, the degree of phosphorylation of STAT3 was augmented. After CTLA-4 expression was up-regulated, T cell activation was remarkably inhibited and the levels of IFN-?, IL-17, IL-22 and other cytokines declined with varying degrees; after small interfering plasmid p GPU6-sh TIRC7 infected target cells, the activation capability of T lymphocyte was enhanced and the secretion of IFN-? and other cytokines was increased.(3) We successfully established the mouse model of moderate and severe acute GVHD. Compared with TIRC7 or CTLA-4 single dose group and non-administered group, the mouse in TIRC7 and CTLA-4 co-administered group had the lowest acute GVHD scores, with the longest average survival time and the shortest recovery time of hematopoietic reconstitution. Starting from 7 day after transplantation, mice liver, lung, intestine and other vital organs in the experimental group developed acute GVHD with different degrees. With the extended transplantation time, the affected organs developed serious acute GVHD pathology, including the gut pathology injury which was peaked on the 14 th day after transplantation, liver and lung pathology damage peaked on the 21 st day after transplantation, and after the above time, the injury degree of organs was gradually reduced. On the 7 day after treatment, there were no significant differences in the pathological lesions of various organs in each experimental group; however, the injury degree of mice liver, lung and intestine in TIRC7 and CTLA-4 co-administered group was amelioated at the 14 th, 21 st, 28 th and 35 th time point. With the extendedtransplantation time, the pathology scores of affected organs in mice with acute GVHD gradually elevated and peaked on the 14 th or 21 st day after transplantation and then gradually declined. The injury degree scores of mice liver, lung and intestine in TIRC7 and CTLA-4 co- administered group were lowest at the 14 th, 21 st, 28 th and 35 th time point.Conclusions:(1) TIRC7 level in patients with acute GVHD was increased and the more severe acute GVHD, the higher level of TIRC7; CTLA-4 level in acute GVHD patients was decreased and the more severe acute GVHD, the lower level of CTLA-4.(2) TIRC7 might positively regulate the function of CTLA-4 and inhibit T cell activation and the secretion of cytokines and then down-regulate the development and progression of acute GVHD.(3) We initially confirmed that when the monoclonal antibody of TIRC7 or CTLA-4 was utilized, the degree of acute GVHD was decreased.