| BACKGROUND AND PERPOSEAllogeneic solid organ transplantation is still the first choice to save the life of patients with end-stage organ failure.Therefore,graft survival is of great importance for prolonging the life of patients.Acute allograft rejection,which was the highest incidence of allograft rejection and the vital factor of graft survival,seriously affects the prognosis of organ transplantation.With the emergence and application of new immunosuppressant,the incidence of acute rejection of allografts has been significantly reduced,and the survival of graft has been prolonged.However,the long-term survival has not been significantly improved,some data showed that it was related with effective therapy of acute rejection.Early detection and early treatment of allograft rejection could significantly improve the survival status of grafts and the prognosis of patients.Therefore,it is necessary to further elucidate the molecular mechanism of allograft rejection and search for the important biomarker tightly related with allograft rejection,to provide an important basis for the early diagnosis of allograft rejection and modulation of immunosuppressive agents' application.T cells recognize the alloantigen,initiate the clone proliferation and differentiation,release a series of pro-inflammatory cytokine,finally result in graft injury and acute allograft rejection.Therefore,how to regulate the proliferation and differentiation of allogeneic effector T cells and how to regulate the inflammatory cytokines related to allograft rejection has become an important means to inhibit acute allograft rejection.Previous studies have shown that CD4+T can directly result in allograft rejection.In Serial Analysis of Gene Expression(SAGE)in allogeneic CD4+T cells and syngeneic CD4+T cells we constructed previously,Stat1 was 10-fold up-regulation in allogeneic CD4+T cells and its upstream IL-27Ra and Ifngr1 was also found up-regulated.Since IFN-y has been proved to promote allograft rejection,but the effect of IL-27R? has not been fully understood,we deduced that IL-27Ra participates in allograft rejection and may be a key molecule to promote allograft rejection.Il-27R is composed of specific subunit IL-27R?(WSX-1)and gp130.Its specific ligand is IL-27,which is composed of EBI3 and specific subunit p28.IL-27 has been proved involved in inflammation,mainly through the modulation of cytokine profile and cell differentiation.On one side,it can activate IL-27R?,release pro-inflammatory cytokines such as IFN-?,IL-6,TNF-? and IL-1?.IL-27 could enhance the antigen processing of dendritic cell,and stimulate the proliferation of CD4+T cells and Th1 type cell response,indicated that IL-27 participates a series of inflammatory response.However,it was reported that IL-27 could promote the secretion of anti-inflammatory IL-10 and reduce airway inflammation.It also inhibited the proliferation of T cells through promoting the function of Foxp3+Treg,and eventually cause anti-inflammatory response.So,the effect of IL-27/IL-27R?pathway on allograft rejection is still unknown.Here,we studied the effect of IL-27Ra on acute allograft rejection and its molecular mechanism,studied its significance as a biomarker of non-invasively imaging acute allograft rejection in vivo.Firstly,the expression and cellular localization of IL-27Ra and IL-27 were studied in the mouse model of allograft rejection.In the second part,we studied the downstream molecular induced by IL-27Ra pathway activation,and explored its molecular mechanism and cellular signal pathway.In the third part,we prepared a radionuclide labeled molecular probe targeting IL-27R?.Through in vivo whole-body dynamic phosphor-autoradiography and targeting analysis,we evaluated whether it can be used as a specific biomarker to early detect the occurrence of acute rejection non-invasively.In the fourth part,a small molecule receptor imaging probe targeting IL-27Ra was prepared by recombinant IL-27,and its ability and characteristics of targeting allograft rejection were studied.The results of this study provided an important experimental basis for elucidating the molecular mechanism of IL-27Ra in allograft rejection,and provided a new strategy for in vivo targeted IL-27Ra radionuclide imaging in monitoring allograft rejection.More importantly,the results of this study are of great significance for targeting IL-27Ra to induce allograft tolerance and prolong graft survival.Part One The expression of IL-27R?+ cells and its effect on inflammatory cytokines during allorejectionMETHOD1.The syn-/allo-skin transplantation models were established and graft survival was analyzed:The skin graft model was established.The recipients were BALB/c mice.C57BL/6 mice and BALB/c mice were used as donor of skin graft for allogeneic and syngeneic transplantation,respectively.The graft was observed daily and survival of graft in different models was analyzed.2.H?E/IL-27R?/IL-27 immunohistochemical staining and Western Blot were performed at different time post transplantation:Allograft and syngraft were separated on day 1,7,10,14,21 post transplantation,and stained with H?E to observe the rejection status and the peak of rejection.The expression of IL-27Ra in acute allograft rejection was confirmed by immunohistochemistry staining and Western Blot.The expression of IL-27 in acute allograft rejection was also confirmed by immunohistochemistry staining and Western Blot.The IL-27 level in graft was detected by ELISA.3.Immunofluorescence staining of IL-27Ra and CD3/CD4/CD68 in grafted skin was performed:On day 10 post transplantation,the skin grafts were separated and double staining of IL-27R?(green light)and CD3 or CD4 or CD68(red light)was performed to confirm the expression of IL-27Ra in CD3+T cells,CD4+T cells and CD68+ macrophage during allograft rejection.4.IL-27R? and CD3/CD4 on recipient spleen were detected by flow cytometry:On day 10 post transplantation,splenocytes were isolated and stained with CD3-PE-Cy7,CD4-FITC and IL-27R?-PE antibodies.The expression of IL-27R?in splenic CD3+T cells and CD4+T cells was analyzed.5.Immunofluorescence staining of IL-27Ra and CD11c/CD3/CD68 in grafted skin was performed:The skin grafts were separated on day 10 post transplantation,and the fluorescent double staining of IL-27R?(green light)and CD11c or CD3 or CD68(red light)was performed respectively to confirm the IL-27 secretion in CD11c+cells,CD3+T cells and CD68+macrophage of the grafts in the allograft rejection.6.Allograft survival after alloreactive IL-27R?+ cells was adoptive transferred to allo-SCID model:The SCID mice model was established(SCID-BALB/c mice were recipients.C57BL/6 mice were donors.No rejection occurred after skin transplantation).After graft healed,the allogeneic splenocytes and IL-27R?+splenocytes sorted by flow cytometry were adoptive transferred.Survival of the allogeneic skin graft in SCID model.At the peak of allorejection,the grafts were separated and stained by H?E staining and IL-27R? immunofluorescence staining to analyze the acute rejection and cell types of local infiltration.7.The analysis of cytokine expression profile:The allogeneic and syngeneic skin grafts on day 10 post transplantation were stained with IL-27R? and IFN-? or IL-10 to analyze IFN-? or IL-10 secretion in IL-27R? positive cells at the time of allograft rejection,and also IL-27 was detected with ELISA.At the same time,the secretion of IFN-y,IL-10 and IL-27 in serum was detected by ELISA to study the change of cytokine profile in the model of allograft rejection.RSULTS1.The expression of IL-27R? in skin grafts at different time post transplantation:The allografts were rejected from day 9 to 13 post transplantation and the syngraft were not rejected.The median survival time was 11 days.Immunohistochemistry staining and Western Blot showed that the expression of IL-27R? was the highest on day 10 post transplantation,significantly higher than that of other time points and the same line control skin grafts.At this time,the expression of IL-27 in allografts was also the highest.2.The expression of IL-27R? on CD3+T cells,CD4+T cells and CD68+macrophage cells.In allogeneic splenocytes,IL-27Ra was expressed on the surface of CD3+T cells,CD4+T cells and CD68+ macrophage cells.On day 10 post transplantation,the proportion of IL-27R?+CD3+T cells and IL-27R?+CD4+T cells derived from allogeneic splenocytes were higher than those of syngeneic splenocytes.3.Secretion of IL-27 by CD3+T cells,CD68+cells and CD11c+cells:On day 10 post transplantation,CD11c+ cells secreted IL-27,while IL-27 was not detected in CD3+T cells and CD68+cells.4.IL-27R? positive cells promoted allograft rejection:Allograft rejection occurred after adopted transferring of alloreactive splenocytes and sorted IL-27R?+ cells to SCID allografted model.The rejection of IL-27R?+cell transferring group was earlier than that of unsorted spleen cells.On day 14 post transplantation,H?E staining and immunofluorescence staining confirmed that much more severe inflammatory cells infiltrated within graft in IL-27R?+ cells adoptive transferred group,which was mainly IL-27R?+ cells.5.The effect of IL-27R? on the expression of IFN-?,IL-10 and IL-27:The secretion of IFN-? and IL-27 was increased and the secretion of IL-10 was decreased in the allografts.In serum,the concentration of IFN-? and IL-27 increased,and the concentration of IL-10 decreased at the top points of allorejection.Part Two Molecular mechanism of IL-27R? activation on allograft rejectionMETHODS1.Mixed lymphocyte reaction(MLR):Splenocytes were isolated routinely the effector cells from spleen of BALB/c mice,and the allogeneic and syngeneic stimulated spleen cells were from C57BL/6 mice and BALB/c mice,respectively.The stimulated cells were pre-treated with Mitomycin,then,mixed with effector cells.2.Effect of rIL-27 on the proliferation of alloreactive splenocytes:Different concentrations of rIL-27 were used to stimulate allogeneic and syngeneic splenocytes for 72h,the cell proliferation was detected with CCK8 method.The splenocytes were stimulated with 25ng/ml rIL-27 for 24h,48h and 72h,then,the cell proliferation was detected by CCK8 method.The splenocytes were treated with JAK inhibitor(Ruxolitinib),STAT inhibitor(SH-4-54)and IL-27 p28 blocking antibody for 72h,then,the proliferation of splenocytes was detected by CCK8 method.3.Effect of rIL-27 on alloreactive splenocytes apoptosis:The expression of anti-apoptotic proteins(Pim2 and Bcl-xL)was detected in alloreactive spleen cells with 25ng/ml rIL-27 stimulation,and treated with 0.12?M Ruxolitinib,1.5?M SH-4-54 and 7.5?g/ml IL-27 p28 antibody for 72h.The total apoptosis of alloreactive splenocytes was analyzed by flow cytometry.4.The effect of rIL-27 on STAT1,STAT3,STAT5 pathway:The mixed allogeneic splenocytes were cultured for 72h.Then,cells were stimulated with 25ng/ml rIL-27 for 0.25h,0.5h,1h,2h and 24h.The phosphorylation and total protein expression of STAT1,STAT3 and STAT5 were detected,and the phosphorylation level was analyzed.The allogeneic splenocytes were cultured for 72h and pretreated with 0.012,0.12,1.2?M Ruxolitinib or 0.15,1.5,15?M SH-4-54 for 24h,then co-incubated with 25ng/ml rIL-27 for 0.25h.The phosphorylation levels of STAT1,STAT3 and STAT5 after JAK/STAT inhibition were analyzed.The alloreactive spleen cells were incubated with 25ng/ml rIL-27,0.12?M Ruxolitinib,1.5pM SH-4-54 and 7.5?g/ml IL-27 p28 antibody for 72h,respectively,to detect the expression of STAT1,STAT3 and STAT5.5.Expression and activation of STAT1/STAT3/STAT5 in allograft at the top period of allorejection:Establishment of syngeneic and allogeneic skin transplantation models.The skin grafts were separated and prepared into paraffin sections on day 10 post transplantation.The expression of STAT1/STAT3/STAT5 was analyzed by Western Blot and immunohistochemistry staining.The immunofluorescent staining of IL-27R?,p-STAT1,p-STAT3 and p-STAT5 was performed to detect the expression of p-STAT1/p-STAT3/p-STAT5 in IL-27R?+cells.RESULTS1.The effect of rIL-27 on the proliferation of alloreactive splenocytes:With the increasing rIL-27 concentration,the proliferation of alloreactive splenocytes apparently increased.rIL-27 promoted the proliferation of alloreactive splenocytes earlier at 24h.After administration of JAK/STAT inhibitor(Ruxolitinib/SH-4-54)and inhibition of IL-27 p28,the proliferation promoting effect of rIL-27 could be obviously down-regulated.2.Effect of rIL-27 on apoptosis of alloreactive splenocytes:rIL-27 inhibited apoptosis of alloreactive splenocytes and promoted the expression of antiapoptotic molecules(Pim2 and Bcl-xL).Inhibition with Ruxolitinib,SH-4-54 and IL-27 p28 antibody reduced the anti-apoptotic effect of rIL-27.3.Effect of rIL-27 on STAT1/STAT3/STAT5 pathway:rIL-27 promoted the phosphorylation of STAT1/STAT3/STAT5 and increased the total protein expression of STAT1/STAT3 and STAT5 in alloreactive splenocytes.4.The expression of STAT1/STAT3/STAT5 in acute allograft rejection:In acute allograft rejection,the expression of STAT1,STAT3 and STAT5 in allograft was significantly increased,and the expression of phosphorylated STAT1/STAT3/STAT5 was detected within IL-27R?+ cells.Part Three Preparation of 125I-anti-IL-27R? mAb and Evaluation its targeting imaging on allograft rejectionMETHODS1.The allogeneic and syngeneic skin transplantation model were established and IL-27Ra expression was analyzed:Establishment of allogeneic and syngeneic skin transplantation model.The recipients were BALB/c mice,and the donors were C57BL/6 mice and BALB/c mice,respectively.The expression of IL-27R? protein was detected at day 6,8,10,12,14 post transplantation.RT-PCR was used to detect the expression of IL-27Ra mRNA in graft.2.The radionuclide probe was prepared and identified:125I-anti-IL-27R? mAband 125I-IgG control were prepared by Iodogen method.The labeling rate,radiochemical purity and stability of 125I-anti-IL-27R? mAb at 24h,48h and 72h in vitro were analyzed.3.The saturation and competitive binding analysis:Single splenocytessuspension was prepared from allogeneic and syngeneic grafted model at day 10 post transplantation(top point of allorejection).The syn-group was used as control.The saturation and competitive binding experiments of 125I-anti-IL-27R? mAb with allogeneic or syngeneic cells were analyzed,respectively.4.Dynamic phosphor-autoradiography:At the peak of allorejection,the specificradioactivity accumulation of 125I-anti-IL-27R? mAb in allografts was observed and semi-quantitative analysis was performed.At the early stage of allograft rejection(day 6 and day 8 post transplantation),125I-anti-IL-27R? mAb specific accumulation in allografts was observed and semi-quantitative analysis were performed.Phosphor-autoradiography was performed for grafted skin and the contra-lateral skin to confirm the specific accumulation of labeled probe.5.Biodistribution study:The biodistribution of 125I-anti-IL-27R? mAb was studiedat day 10 post transplantation,and the target to non-target ratio(T/NT ratio)was calculated.Meanwhile,biodistribution was also analyzed at the early stage of allorejection(day 6 and day 8),and the T/NT ratio was calculated.6.Correlation between the uptake of 125I-anti-IL-27R? mAb probe and theexpression of IL-27R?:The radioactivity ratio(the DLU/mm2 rate of graft and opposite normal skin)of allogeneic and syngeneic models on day 6,8,10 post transplantation and the expression of IL-27R? protein of skin grafts at the same time were analyzed.RESULTS1.The expression of IL-27R? during allograft rejection:At the protein level,the highest expression of IL-27R? was found at the peak of allograft rejection(day 10).And also,higher IL-27R? expression was observed than that in syngeneic grafts at the early stage of rejection(day 6 and day 8),At the mRNA level,the expression of IL-27R? in allografts was higher both than that in syngrafts and control skin.2.Preparation and identification of 125I-anti-IL-27R? mAb and 125I-IgG:The radiochemical purity of 125I-anti-IL-27R? mAb and 125I-IgG were 96.87%and 96.86%respectively.The stability was kept up to 90%until 72h.3.Saturation and competitive binding analysis between 125I-anti-IL-27R? mAb and splenocytes:Bmax value of syngeneic spleen cells was 2041 cpm/106 cells.Kd value of syngeneic spleen cells was 13.09nM.The Bmax value of the alloreactive splenocytes was 4824 cpm/106 cells,and the Kd value was 13.84nM.The specific binding of 125I-anti-IL-27R? mAb to alloreactive splenocytes was almost totally blocked by unlabeled IL-27R? antibody adding.4.Dynamic phosphor-autoradiography:At the peak period of allorejection,the imaging at 24h,48h and 72h showed that allograft accumulated higher radioactivity in group of 125I-anti-IL-27R? mAb injection than that of 125I-IgG control group and blocking group,and the radioactivity accumulation of 125I-anti-IL-27R? mAb could be blocked by pre-injecting unlabeled anti-IL-27R?mAb.At 48h,the radioactivity of allografts was significantly higher than that of the control skin.At the same time,the imaging was the clearest and the contrast was high.And interestingly,in the early stage of allorejection(day 6 and 8 post transplantation),the accumulation of 125I-anti-IL-27R? mAb in allografts was also higher than that in syngrafts.5.Biodistribution study:The 125I-anti-IL-27R? mAb was mainly metabolized through liver and kidney.The uptake of 125I-anti-IL-27R? mAb in allogeneic skin graft was the highest,significantly higher than that in syngeneic skin graft and control skin.The T/NT ratio of the allogeneic group was significantly higher than that of the syngeneic group,125I-IgG group and blocking group.6.Correlation of 125I-anti-IL-27R? mAb uptake and IL-27R? expression in skin graft:The radioactivity ratio of allogeneic and syngeneic models on day 6,8,10 post transplantation was high-positively correlated with IL-27R? protein expression of skin grafts at the same time points(r=0.9719,P<0.01).Part Four Preparation of IL-27R?-targeted radionuclide-labeled probe and evaluation on monitoring allograft rejectionMETHODS1.Established the allogeneic and syngeneic skin transplantation mice model following the same method in the third part.2.The radionuclide probe was prepared and identified:125I-rIL-27 was prepared by Iodogen method.The labeling rate and radiochemical purity were analyzed.The stability of 125I-rIL-27 at 1h,12h and 24h was analyzed.Lipophilicity was detected.3.Saturation and competitive binding analysis:Spleen were isolated at the peak of allogeneic transplantation(day 10 post transplantation),and the spleen of the syngeneic transplantation group used as the control.The single cell suspension was prepared.The competitive binding and saturation of 125I-rIL-27 with splenocytes were analyzed.4.Dynamic phosphor-autoradiography:After injection of 125I-rIL-27,whole body dynamic phosphor-autoradiography for model mice and skin grafts in groups of allogeneic and syngeneic grafted mice were performed,also,blocking group by pre-injection with unlabeled anti-IL-27R? mAb in allografted group was studied.Semi quantitative analysis was performed.5.Biodistribution study:Biodistribution ex vivo was analyzed at 24h,and T/NT ratio and T/B ratio were calculated.6.Blood clearance of labeled probe:After 125I-rIL-27 and 125I-anti-IL-27R? mAb injection,the blood clearance analysis was performed and calculated.7.Correlation between the uptake of 125I-rIL-27 probe and the expression of IL-27R? in graft:Correlation analysis between the radioactivity(DLU/mm2)of allogeneic and syngeneic skin graft and the expression of IL-27Ra protein was performed.RESULTS1.Preparation and identification of 125I-rIL-27:The radiochemical purity of 125I-rIL-27 was 93.28%.The stability was kept up to 90%until 24h.The 125I-rIL-27 probe was hydrophilic.2.Competitive binding and saturation analysis of 125I-rIL-27 with splenocytes:Bmax value of syngeneic spleen cells was 1607 cpm/106 cells,and Kd value was 49.04 nM.Bmax and Kd of the allogeneic spleen cells were 2545 cpm/106 cells and 48.59 nM,respectively.Unlabeled anti-IL-27Ra mAb could block the specific binding of 125I-rIL-27 to alloreactive splenocytes.3.Dynamic phosphor-autoradiography:At 6h after injection,obvious radioactivity accumulation could be observed in allograft,and much more radioactivity accumulated in allograft than syngraft at 24h.Pre-injection of unlabeled anti-IL-27R? antibody could almost totally block the radioactivity accumulation in allografts.Compared with 125I-anti-IL-27R? mAb imaging,the background of 125I-rIL-27 injection group was much lower.4.Biodistribution study:The uptake of 125I-rIL-27 was the highest in the allografts at 24h,significantly higher than that in the allografts and the control skin.The T/NT ratio and T/B ratio in the allo-transplantation group were significantly higher than those in the syn-transplantation group.5.Blood clearance assay:Compared with 125I-anti-IL-27R? mAb injection group,125I-rIL-27 injection group showed shorter blood retention time and faster blood clearance.6.Correlation between the uptake of 125I-rIL-27 and the expression of IL-27R?in graft:There was a highly positive correlation between radioactivity and the expression of IL-27Ra protein in the same period both in allogeneic and syngeneic skin grafts(r=0.7169,P<0.05).CONCLUSIONS1.IL-27Ra was highly expressed in the graft and spleen during acute allograft rejection,and the expression cells were mainly CD3+T cells and CD68+macrophages.Adoptive transfer of IL-27R?+cells accelerate allograft rejection.2.CD3+IL-27R?+cells and CD68+IL-27R?+cells induced IFN-? secretion and inhibited IL-10 secretion.CD11c+cells secreted IL-27.3.Activation of IL-27Ra could up-regulate the proliferation of alloreactive spleen cells,increase the expression of anti-apoptotic molecules and inhibit apoptosis of alloreactive spleen cells through STAT1/3/5 pathway.4.125I-anti-IL-27R? mAb was successfully prepared,which could specifically bind to IL-27R? on alloreactive spleen cells in vitro and could specifically target to the allografts in vivo.It could be used in monitoring the acute rejection of allografts in the early stage.5.The small molecule receptor targeting probe(125I-rIL-27)was successfully prepared and could specifically bind to the IL-27R? on alloreactive spleen cells in vitro,and accumulated with allograft at the top period of allorejection in vivo,with the fast blood clearance and low imaging background,may be used in specifically monitoring the acute allorejection.INNOVATIVE POINTS1.IL-27R? is highly expressed in rejecting allografts,and adoptive transfer of IL-27Ra positive cells promoted allograft rejection.2.The mechanism of IL-27R? positive cells promote allogeneic rejection mainly through increasing the expression of IL-27R? in CD3+T cells and CD68+macrophages,increasing the expression of anti-apoptotic protein,enhancing STAT1/3/5 phosphorylation,promoting IFN-? secretion and reducing IL-10 secretion.3.Two kinds of radioisotope labeled probes targeting IL-27R? were successfully prepared.Two probes could both specifically target the rejecting allograft and could be used non-invasively monitoring allograft rejection.And more importantly,radioactivity accumulation in graft were highly positively correlated with the expression of local IL-27R?,even in early stage of allorejection,which indicated that IL-27R? could be used as a molecular target for early detection of acute allorejection. |
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