Impact Of CDK9 Inhibition For T Cells On TLR5-Targeting Monitoring During Allorejection

[Background and Purpose]As no effective alternative treatment is applied to the therapy of end-stage organ failure till now,transplantation becomes to the only option to save the lives of patients.However,the immunological rejection of grafts plays the key role during allorejection.Allograft rejection,a complex immune response process,involves several immune-related cytokines mediated immune injury mechanism.The main causes of that is the difference of HLA between recipients and grafts.With the widespread application of immunosuppressive therapy,the survival rate of graft and the quality of life of the patients are increasing However,with the lack of selectivity and specificity of immunosuppressant not only abnormal,but also normal immune response can be suppressed.It may induce the declining in resistant to organisms.What's worse,severe infection and malignant tumors is becoming so as to influence the long-term survival rate of grafts.Therefore,the dynamic grafts monitoring will be a great significance to early diagnosis and treatment supplement of acute allograft rejection.In clinical practice,the allorejection monitoring methods is abundant,such as clinical features,biochemical indexes in peripheral blood,pathological biopsy of graft and image logical diagnosis currently.Pathological biopsy is the most reliable means of all,however,the invasiveness and potential complications limiting its clinical application.The lack of sensitivity and specificity of other ways makes it difficult to detect the lesions in early stage.Consequently,what becomes a crucial problem in the area of transplantation immunology is to explore non-invasive monitoring target molecules in order to dynamic monitor the allograft rejection effectively.To be a kind of pattern recognition receptors,Toll-like receptor family(Toll-like receptor,TLRs),has a special recognition in pathogen-associated molecular patterns(PAMPs).Recent researches have shown that TLRs express on the surface of dendritic cells and macrophages extensively,T lymphocytes certainly.The only negative regulator of immune molecules in TLRs family,TLR5,whose only protein ligand is flagellin,can participate in the pathogenesis process of autoimmune disease and malignant tumor.It was also shown that Flagellin-TLR5 activated the access of NF-KB,the associated proinflammatory cytokines of which secreted.It has also been reported that TLR5 is highly expressed on regulatory T? cells and participates in immuno-negative regulation.The studies of our previously work indicated that TLR5 has high-expression in the allograft rejection peck period.The application of rapamycin induced allograft tolerance,but it could promote the expression of TLR5 in the graft site.Results suggested that the expression of TLR5 is closely related to alloimmune tolerance,which is likely to be the target point in dynamic monitoring allograft rejection.Unfortunately,some studies reported that toxic and side effects of existing immunosuppressive agents like rapamycin,resulted in liver,kidney and other important organ damage.Hence,it is urgent to explore new drugs to inhibit allograft rejection.CDK9(Cyclin-dependent kinase 9),a component of the positive transcription elongation factor b(p-TEFb)kinase,phosphorylates the RNA polymerase II to start transcription.A large quantity of researches showed that CDK9 played a vital role in motoring initial inflammatory gene activation and controlling inflammatory process.In terms that the essence of the allograft rejection is inflammatory reaction and CDK9 is closely related to inflammation,we predict that CDK9 inhibition is likely to influence the expression of on the T cells of allograft rejection.As a result,this paper choose the allograft acute rejection model,labeled anti-TLR5mAb with radionuclide iodine-125,explored the impact of CDK9 inhibition on TLR5 expression in order to prove that TLR5 could be the motoring allorejection molecular target and regulating T cells activity and to provide a new basis for motoring allorejection.In this thesis,the effects of CDK9 inhibition for T cells on TLR5-targeted monitoring of allograft rejection are investigated in two parts.Part one,the effect of CDK9 inhibition on the expression of TLR5 of allo-reactive lymphocytes was investigated in vitro;Part two,the effect of T cells CDK9 inhibition on TLR5-targeted monitoring of allograft rejection is investigated in vivo.Part one Effect of CDK9 inhibition on TLR5 expression in allogeniceffector cells and preparation of 125I-anti-TLR5 mAb[Methods]1.CCK-8 allogeneic spleen cells proliferation assaySpleen cells were prepared from BALB/c mice used as effector cells and from C57BL/6 mice used as stimulated cells,divided into Control group and CDK9 inhibition group(CDK9 inhibitor,LDC000067 treatment)and proliferation of cells was detected with CCK-8 assay.2.Western blotThe cells treated with different concentrations of the agent(LDC000067)to detect the expression of CDK9 in the allogenic spleen cells,and combined with cell proliferation assay to obtain the optimal concentration of CDK9 inhibition.The expression of TLR5 in allogenic spleen cells was detected at the optimal concentration of CDK9 inhibition.3.Preparation of 125I-anti-TLR5 mAb 125I-anti-TLR5mAb was prepared with Iodogen method and purified,the label rate was calculated.4.Radiochemical purity and stability of 125I-anti-TLR5 mAbThe radiochemical purity and Rf value of 125I-anti-TLR5 were measured.Mixed protein peak of labeled agent with NS,Serum separately,and measured the stability of 125I-anti-TLR5 mAb.5.Ratio of cell uptake and dissociation with 125I-anti-TLR5 mAbAllogenic effector cells were prepared from BALB/c mice,divided into Control group and CDK9 inhibition group,incubated 6 hours,and adjusted the cells concentration.Then the allogenic spleen cells and 125I-anti-TLR5 mAb were mixed and incubated at the time of 0.25,0.75,3,6,24 hours,ended by the use of pre-cooling PB.The cells were collected for measuring the radioactivity and calculating the ratio of cell uptake.The cell preparation and agent treating of the dissociation experiment was carried just the same as above.The cells were incubated 24 hours,washed,then resuspended with RPMI-1640 culture medium and continuously incubated until the time of 0.25,0.75,3,6,24h by the use of pre-cooling PB.Supernatant is acquired and measured the radioactive counts for calculating the ratio of cell dissociation.[Results]1.Effect of CDK9 inhibition on proliferation of allogeneic spleen cellsThe results showed that different concentrations of CDK9 inhibitor could inhibit effector cell proliferation,and the optimal concentration for inhibiting effector cell proliferation was 5?M(P<0.05).2.The effect of CDK9 inhibition on the expression of TLR5 in allogeneic spleen cellsThe results showed that after treatment of effector cells with different concentrations of CDK9 inhibitor,the expression of CDK9 was reduced to a different extent compared to the control group,and the expression of CDK9 was the lowest when the concentration was 5 ?M.According to the results of cell proliferation,the best CDK9 inhibitory concentration was 5?M,and the expression of TLR5 was increased at this concentration compared with the Control group(P<0.05)apparently.3.Label rate,radiochemical purity and stability of 125I-anti-TLR5 mAb125I-anti-TLR5 mAb was prepared successfully with 96.2%high labeled rate and 0.11 in Rf value.It also had high stability in NS and serum which showing no significant difference between these two groups,the value of which still maintained above 90%until at 72 hour.4.Ratio of Cell uptake and dissociation with 125I-anti-TLR5mAbThe uptake ratio of 125I-anti-TLR5mAb in two groups appeared to be an increasing trend.However,the inhibition group was higher than the control group at 0.5 to 24hours.Except for 0.25 hours,there were significant differences were detected between the two groups at the other 4 time points.The ratio of dissociation with 125I-anti-TLR5mAb was increased with the time passing,the ratio of inhibition group lower than that of control one,which had a statistical difference at 0.25,0.75 and 24h.Part two In Vivo Studies of CDK9 inhibition for effector T cells on TLR5-targeting monitoring during allorejection[Methods]1.Establishment of C57BL/6-BALB/c SCID skin allograft mice modelAllograft models were established with donor C57BL/6(B6,H-2b)female mice and recipient SCID(H-2d)female mice according to the standard methods.7 days after surgery,unpacking,skin graft was observed daily.3 weeks after transplantation,the growth of black hair on the graft site was observed which mean the allo-transplantation model successfully established.2.Isolation of BALB/c mice T cells and groupingIsolating BALB/c mice T cells according to the standard methods,and purified with mice T Cell Enrichment Column.Then T cells treated with LDC000067(5?M)or equivalent PBS were collected and resuspended,adoptive transferred into C57BL/6-SCID model mice separately,which were divided into control group and CDK9 inhibition group.3.Allograft survival with CDK9 inhibition of T cellsThe graft of two groups mice adoptive transferred into T cells was observed and recorded daily until completely allorejection occurred in Control group.4.Biodistribution in allotransplantation model of 125I-anti-TLR5mAbThe two groups were injected with 125I-anti-TLR5mAb through tail vein after thyroid blocked at the peak of control group rejection separately.Animals were sacrificed after 72 hours,collected blood,grafts,opposite normal skin and other important organs,tissues to study the biological distribution and calculate T/NT(Target/Nontarget,T/NT ratio)in two groups of mice.5.Dynamic whole-body phosphor-autoradiography in allograft mice modelThe model mice were injected with 125I-anti-TLR5 mAb through tail vein after thyroid blocked at the time of rejection peak of control group.The specific block group selected from the inhibition group was injected with the unlabeled anti-TLR5mAb.Dynamic in vivo whole-body phosphor-autoradiography was obtained at 24h,48h,72h for image analysis.6.Histological staining and CDK9/TLR5 expression by immunohistochemical stainingSkin grafts from the two groups of animals which have been performed dynamic whole-body phosphor-autoradiography were harvested to be fixed into 4%paraformaldehyde,embedded into paraffin,sliced for HE and CDK9/TLR5 immunohistochemical staining.[Results]1.C57BL/6-BALB/c SCID skin allograft mice model were established successfullyGrafts were soft,moist,no granular sensation,swelling and exudates at the day 7 after transplantation.Black hair is growing at the week 3 after transplantation,which is verified the success of C57BL/6-SCID skin allograft mice model.There was no adverse reaction after the T cells adoptive transferring,the result confirming the success of T cells mediated C57BL/6-SCID skin allograft mice model.2.Allograft survival with CDK9 inhibition for T cellsT cells CDK9 inhibition apparently prolonged allograft-survival from 20±1.58d to 29±2.77d by the means of observing the mice grafts.There is a statistical difference between the two groups.It is suggested that CDK9 inhibition can significantly prolong the survival of allograft.3.Biodistribution in allotransplantation model of 125I-anti-TLR5 mAbLiver,kidney and grafts were high radioactive counted in the two groups.The counts of inhibition group grafts were evidently higher than control group,T/NT ratio(allograft skin/opposite skin)of CDK9 inhibition group higher than that of control group(3.70±0.16 vs.2.02±0.06,which has statistical difference.4.Dynamic whole-body phosphor-autoradiography in allograft mice model The two groups showed obvious grafts imaging after 48,72h of injection with 125I-anti-TLR5 mAb,which was consistent with the result of biodistribution.Inhibition group showed much clear than control group.Block group grafts had no developing.There was an interaction between groups and time,and the difference between the three groups was statistically significant(P<0.05).The radioactivity of 48h inhibition group was higher than control(2.35±0.19 vs.1.11±0.09,P<0.05);Inhibition group(2.09±0.01)and block group(1.09±0.04)compared with control one showed a evident statistical difference until 72h(P<0.05).Phosphor-autoradiography imaging results are consistent with the biological distribution in vivo.5.Histological staining and CDK9/TLR5 expression with immunohistochemical stainingCompared with the Control group,the Inhibition group grafts was weaken inflammation and increased expression of TLR5 on allograft according to the results of histological and immunohistochemical staining.[Conclusion]1.CDK9 inhibitor can significantly inhibit the proliferation of alloreactive effector cells and promote the expression of TLR5.Inhibiting the allotransplanted T cells CDK9 can significantly prolong the survival of allograft;the inflammatory infiltration of allografts is significantly reduced,while the expression of TLR5 is increased.2.The 125I-anti-TLR5 mAb was successfully prepared.The inhibition of CDK9 on spleen cells significantly promoted the affinity of TLR5 in the splenocytes with 125I-anti-TLR5 mAb.CDK9 inhibition of T cells promoted the localization of allograft with 125I-anti-TLR5.[Points of innovation]1.Expression of TLR5 on alloreactive spleen cells was increased with treatment of CDK9 inhibitor LDC000067.2.CDK9 inhibition of T cells promoted the localization of allograft with 125I-anti-TLR5.The results suggest that inhibition of CDK9 is beneficial to the monitoring of TLR5-targeted allograft rejection in vivo.