Study Of Bcl-6 Downregulation To Prevent Chronic Graft Versus Host Disease

Background and Objectives:Chronic graft versus host disease(cGVHD)is a kind of chronic inflammation and fibrosis pathological damage of multiple organs and tissues.The clinical feature is similar to the syndrome of autoimmune disease.cGVHD is the main complication causes death after the allogeneic hemapoietic stem cells transplantation(allo-HSCT).As allo-HSCT are increasingly used in older patients,as well as an increasing number of the haploid transplantation and donor lymphocyte infusion,an upward trend appears in the incidence of cGVHD.Although we have produced a large number of new immunosuppressant,in addition to the cortical hormone as a first-line therapy,other drug curative effect is poor.And refractory cGVHD case fatality rate is as high as 50%.Existing immunosuppressive therapy is given priority to unselective inhibition of T cell or remove B cells.The untargeted treatments lead to malignant tumor relapse,severe infection,secondary tumor and increasing cGVHD again and again,which seriously affects life quality of patients.Therefore,further explore of the pathogenesis of cGVHD and the precise mode of targeted therapy has the vital significance in the field of transplantation immunity research.Human cGVHD clinical manifestation is similar to systemic lupus erythematosus(sle),myasthenia gravis,sjogren's syndrome and other autoimmune diseases.But its pathogenesis is still unclear.A large number of studies have shown that B cells are antigen presenting cells which launch specific T cells' immune response and are the effector cells of the humoral immune system.They are playing a more and more important role in occurrence and development in cGVHD.And B cell activation and function implementation depends on the auxiliary T cells.Recent studies have found Follicular Helper T cells(Tfh),located in lymphoid follicles,expressing CXCR5,inducible costimulatory(ICOS),B-cell CLL/lymphoma 6(Bcl-6),PD-1 and IL-21,is the main auxiliary cells of B cells and play a key role in the start the humoral immune response.Microenvironment signals stimulus(such as IL-21)and STAT3 activation strengthen Tfh cells ability of B cell auxiliary function.Both animal experiments and clinical preliminary studies confirmed that the excessive activation of Tfh cells and the related abnormal molecular expression can cause the increasing antibody generation of plasma cells,the disordering of antibody mediating humoral immune response.And it finally cause autoimmune disease.However,what's Tfh's role in cGVHD characteristic of autoimmune diseases?Inland and abroad research about this is poor.Through mouse spleen cells infusion,Foster AD and Nguyen V found in lupus-like cGVHD mice model that high expression of ICOS and IL-21 in CD4+T cells is closely related to the severity of the disease.A large number of studies have shown that the Bcl-6 is the key transcription factor to regulate the initial T cells'differentiation into Tfh.When Naive initial CD4+T cells differentiation into each subgroup,the Bcl-6 preferentially expression on Tfh by STAT3 pathway,but not Th1 or Th2 cells.The expression of CXCR5 depends on the Bcl-6,The lack of the Bcl-6 T cells cannot be differentiated into Tfh cells.The Bcl-6 can effectively protect the precursor B cells' Ig-affinity-mature process in the germinal center from apoptosis,and can cause the differentiation into plasma cells,and antibody production,futher.Animal experiments have demonstrated that the Bcl-6-defect mice are unable to form a germinal center(GC)or reduce GC formation.When cGVHD progress in patients,our early researches find that,CD19+CD5+B cells decrease and CD 19+CD5-increase.And the B cell subsets disorder can be recovered along with the improvement of the disease.And expression profile chip showed an increasing of the Bcl-6 adjustment factor of STAT3 and IL-21 R expression level in patients with cGVHD.The Bcl-6 gene is the specifically transcriptional switch for the differentiation,proliferation and function implementation of Tfh.IL-21 is one of the important regulatory factor of Tfh and B cell proliferation.On the latest research progress and previous research,we proposed that Tfh participate in the pathogenesis in cGVHD and specifically block the Tfh control factor,Bcl-6.This will be a scientific hypothesis that new targets for cGVHD prevention and control.We proposed to establish a cGVHD model of specific blocking the Bcl-6,and detecting the Bcl-6 gene expression level and quantity of the secretion-IgG-antibody cells in GC.Thereby,we can explore the role of Bcl-6 regulation Tfh in cGVHD and its related mechanism.This provides the experimental basis for the cGVHD targeted therapy,which has great theoretical and practical value.Methods:1.To establish a cGVHD mice model like the occurrence and development of clinical cGVHD.BALB/C female mice were divided into blank group,BMT group,TBI group and cGVHD group.BALB/c female mice after the linear accelerator 700 cGy of whole body irradiation(TBI),accepted major histocompatibility(MHC)antigen matched,minor histocompatibility antigens(miHA)mismatched of B10.D2 male mice spleen cells and bone marrow cells(8 × 106,1:1)by tail intravenous,can build a cGVHD model like human cGVHD.After TBI,BMT group accepted B10.D2 bone marrow cells(8 × 106),while TBI group accepted nothing after TBI.By observation of peripheral blood leukocyte count and chromosome of mice after transplantation to verify hematopoietic reconstruction and implantation.After transplantation weighted mice every day.14 d after transplantation to observe the clinical features of mice,like body rash,hair removal,a carpenter,diarrhea,etc.,every 3 days.Analyzed clinical cGVHD score between BMT group and cGVHD and evaluated target organ pathological changes at the end point of observation after transplantation.2.15 mice with randomly divided into blank group and DMSO group and 79-6 group according to weight at 12th d after transplantation.79-6 group:five mice were accepted daily intraperitoneal injection with 79-6 by 50 mg/kg for 5days(injection volume according to the weight of mice,10 ul/1 g);DMSO control group:each mice was accepted daily intraperitoneal injection with 10%DMSO for 5 days(injection volume according to the weight of mice,10 ul/1 g);Blank group:each mice were accepted daily intraperitoneal injection with PBS for 5 days(injection volume according to the weight of mice,10 ul/1 g).Observed the clinical features after treatment:sacrificed the mouse if the weight less than 13 g or end point of observation.Target organs were harvested for the test as following:(1)target organs like skin,lung,liver,etc for HE stainning in order to analysis the cGVHD score.(2)compare the cGVHD clinical grading,survival time of three groups.(3)RNA of peripheral blood were extracted and quantitative real-time PCR were performed to detect mRNA levels of Bcl-6 gene.(4)spleen paraffin sections were used for immunohistochemical stainng of IgG.(5)spleen tfh cells measured by flow cytometry.2.Statistical analysis was performed by SPSS 19.0 software.The One-Way ANOVA was used to compare multiple sets of datas,LSD analysis was performed to the comparison of single factor analysis between two groups.Two sets of measurement data were compared by statistical analysis using two independent samples t test.Different time for measurement of samples,using the variance analysis of repeated measurement design.Kapplan-Meier method was used to analysis the survival time.All statistics were 2-tailed,and siginicance was set at P<0.05.Results:1.The experiment constructed a cGVHD murine model with MHC-matched miHA-mismatched B10.D2?BALB/C.In the 10th day after the transplantation,all the mice in the blank group,the transplantation group and the cGVHD group were survived.Their white blood cell counts were greater than 1×109/L,which affirmed their recovery of hematopoiesis.Meanwhile,the mice in the TBI group were dead of hematopoiesis failure.The median time of death was 10 days.The mice did not dead in the transplantation group and the cGVHD group until the end of our observation.Moreover,the cGVHD clinical scores of the mice in the cGVHD group were higher than that of the mice in the transplantation group,which showed the statistical significance(F=53.59,P=0.00).After the 20th day after transplantation,the skin's clinical scores of the all mice in the cGVHD group were over 0.6.The target organ,such as skin,liver,lung,of them emerged typical cGVHD pathological changes.This showed the statistical significance between the mice in the cGVHD group and other control groups(P<0.05).2.In the 14th day after the transplantation,swell,decrustation and incrustation could be found on all the mice's tails,claws and ears on both sides.In the 30th day after the transplantation,which was the 18th day after the beginning of cure,swell,decrustation,incrustation,anabrosis and sclerosis could be found on the tails,claws and ears on both sides of the mice in the blank group and the DMSO control group.And unhairing skin was also found on the back and neck of them.In the meantime,it could be found that the unhairing skin on the back and neck of the mice in 79-6 treating group had a smaller area.Along with the time lapse,reviviscence,gaining weight and alleviated symtoms,such as skin incrustation,sclerosis,unhairing and anhelation,were observed on the mice in 79-6 treating group.While the mice in the blank group and the DMSO control group appeared to be cachexia,weight loss,growing of skin's unhairing and incrustation,anhelation and arched back which caused dyskinesia.And their cGVHD clinical score maintain in a high level.All the mice in the DMSO control group were dead or killed at the end point of observation.(1)The results of ANOVA for repeated measurement analysis of the mice's cGVHD clinical scoring showed that time effect was statistically significant(F=3.559,P=0.006),which illustrate that cGVHD mice clinical scores changed over time.Time and our intervention interaction effect was also statistically significant(F=2.361,P=0.043).The trend of change of the cGVHD clinical scores grew over time in the 79-6 treated group and two control groups' mice.Kapplan-Meier survival analysis showed that the blank control group mice expected an average survival time for 50±13.14 days,while the DMSO control group mice expected an average survival time for 44±3.29 days,the 79-6 treated group mice expected an average survival time for 62±5.57 days.The survival time of DMSO control group and 79-6 treated group had significant difference(P>0.05).(2)The relative expression of Bcl-6 mRNA in the peripheral blood of the mice in blank control group,DMSO control group and 79-6 treated group had significant difference(F=23.40,P=0.000).Its expression quantity of 79-6 treated group mice was lower than that of DMSO control group mice(t=5.89,P=0.001),and lower than the blank control group(t=5.431,P=0.002).And the differences were statistically significant.(3)The one-way ANOVA analysis with target organs' pathological score of mice in all groups with showed that total pathological scores difference were statistically s ignificant between all groups(F=5.57,P=0.027).The 79-6 treated groups' Bcl-6 mR NA expression was lower than the DMSO control group(P<0.05),lower than the bl ank control group(P<0.05).Their difference had statistical significance.According todifferent target organs,the analysis showed as follow.The difference of skin patho logical score in each group mice was statistically significant(F=5.46,P=0.028).The 79-6 treated group mice's skin pathological scores were significantly lower than that of the blank control group mice and DMSO control group mice(P<0.05).The lung pathological score of 79-6 treated group mice had no obvious advantage compared wi th the two control group mice(P>0.05).(4)According to the preliminary results of immunohistochemical IgG stainning,the spleens of mice in blank control group and DMSO control group mice had IgG positive cell cytoplasm dense germinal centers.The spleens of mice in 79-6 treated gr oup mice had significantly decreased IgG-positive cell germinal centers.(5)There was no significant difference of spleen Tfh in 79-6 group,compared with blank group and DMSO group.Conclusions:1.Successfully established B10.D2?BALB/C cGVHD mouse model,with the feature of inflammation,collagen fibrosis of the target organ like skin,lung,gastrointestinal tract,liver and other organs.Providing a good mice model to further study the pathogenesis and treatment of cGVHD.2.The Bcl-6 small molecule inhibitors 79-6 cGVHD mice in vivo treatment achieved initial curative effect,clinical feature like scabby recovered,weight gain;the feature of inflammation,collagen fibrosis of the target organ like skin,lung,was less serious;the ability of B cell antibody secretion declined.Downregulated Bcl-6 can prevent cGVHD.