Study On PD-1 Mediated Immune Tolerance Via T Cells In Alveolar Echinococcosis With Clinical And Experimental Researches

Objective:Alveolar Echinococcosis(AE)is called by“parasitic cancer”because of the characteristic of progressive growth and budding proliferation.Many studies have shown that the suppressive signals which included in the secondary signal participated the interplay with the hosts and Echinococcus multilocularis(E.multilocularis),affecting the immune responses,the result of location and the course of the disease from the startand development to transportation.The aims of the study:1)To investigate the mechanism of the immunosuppressive molecules programmed death receptor-1(PD-1)mediated the immune tolerance of AE and the mice model of E.multilocularis via T cells;2)To investigate the effect and mechanism of blockade of PD-1/PD-L1 signaling pathway in the mouse model infected with E.multilocularis,the possibility of new immune target therapy in the E.multilocularis infection.Methods:Part one,The C57BL/6 mice model was established with the protoscoleces(PSCs)of Echinococcus granulosus(E.granulosus)via portal vein.Using the histological technique and flow cytometry(FCM)to compare the histology and immune reaction between C57BL/6 mice infected with E.granulosus and C57BL/6 mice infected with E.multilocularis.Part two,1)Liver specimens of 20 patients with AE were collected after operation,and the lesion and changes surrounding the lesion were detected with pathological tests;clinical data of these patients were collected and detected the liver histological changes;immunohistochemistry(IHC)and Masson staining were used to detect the fibrosis of liver.2)FCM was used to detect the immune cells surrounding the lesion and the expression of PD-1 on T cells;q RT-PCR was used to detect the expression of PD-1 and analyzed the relation of hepatic function;3)flow cytometry was used to compare the cytokines secreted from the positive PD-1 and negative PD-1 of T cells.Part three,1)The BALB/c mice model was established with the PSCs of E.multilocularis via portal vein,separated the lymphocyte from the liver,detect the expression of PD-1 on T cells with the flow cytometry and compared the cytokines secreted from the positive PD-1 and negative PD-1 of T cells.2)BALB/c,Nude and SCID mice models were established with the PSCs of E.multilocularis via portal vein,observed the macroscopic changes and test the related data,compared the percentage of infection,the location of lesion and the number and volume of lesions.The pathological technology and staining were used to compare the histopathological alterations.Sirius red staining and immunohistochemistry were used to detect the fibrosis of liver.3)C57BL/6 mice models were established with the PSCs of E.multilocularis via portal vein,and then injected the anti-CD4⁺T cells antibody,anti-CD8⁺T cells antibody and the isotype antibody.After 12weeks,the changes of liver were observed and measured,and compared the percentage of infection,the location of lesion and the number and volume of lesions between 2 groups.The pathological technology and staining were used to compare the histopathological alterations.Part four,1)BALB/c and the PD-1 knockdown(PD-1^-/-)mice models were established with the PSCs of E.multilocularis via portal vein,and the changes of liver were observed and measured,and compared the percentage of infection,the location of lesion and the number and volume of lesions between 2 groups.The pathological technology and staining were used to compare the histopathological alterations.Sirius red staining and immunohistochemistry were used to detect the fibrosis of liver.Separating the lymphocyte from the liver,detected the number of lymphocytes,the percentage of activated T cells,the percentage of the subgroups of memory T cells and the subgroups of T cells(Th1,Th2,Th17 and Treg)in the liver infected with E.multilocularis.With the flow cytometry and compared the cytokines secreted from the positive PD-1 and negative PD-1 of T cells.BALB/c and the PD-1 knockdown(PD-1^-/-)mice models were established with the PSCs of E.multilocularis via subcutaneous injection,and the changes of liver were observed and measured,and compared the percentage of infection,the location of lesion and the number and volume of lesions between two groups.The pathological technology and staining were used to compare the histopathological alterations.2)C57BL/6 mice models were established with the PSCs of E.multilocularis via portal vein,and then injected the anti-PD-1 antibody,anti-PD-L1 cells antibody and the isotype antibody after 12 weeks post-infection.The changes of liver were observed and measured,and compared the percentage of infection,the location of lesion and the number and volume of lesions between 2 groups.The pathological technology and staining were used to compare the histopathological alterations and detected the fibrosis of liver.Separating the lymphocyte from the liver,spleen,the lymph node and vein blood,detected the number of lymphocytes,the percentage of activated T cells,the percentage of the subgroups of memory T cells and the subgroups of T cells(Th1,Th2,Th17 and Treg)in the mice infected with E.multilocularis with the flow cytometry and compared the cytokines secreted from the positive PD-1 and negative PD-1 of T cells.Results:Part one,1)CD4⁺T cells and CD8⁺T cells increased at early stage in mice model infected with E.granulosus,but at middle and late stage in mice model infected with E.multilocularis.Part two,1)‘immune inflammatory microenvironment'surrounding the lesion was existed in AE patients,with characteristic of fibrosis and hepatic function impairment.2)T cells surrounding the lesion included CD4⁺T cells and CD8⁺T cells,with macrophages and other cells.3)PD-1 was expressed on the T cells and was higher in the close liver tissue than the distant liver tissue,and the expression of PD-1 on the CD4⁺T and CD8⁺T cells were higher in the close liver tissue than the distant liver tissue(21.11±1.15 vs11.69±0.69%,22.31±1.15 vs 11.42±0.79%,P<0.05).Secretion of IFN-?and TNF-?from PD-1^-CD4⁺T and PD-1^-CD8⁺T cells was higher than PD-1⁺CD4⁺T and PD-1⁺CD8⁺T cells in close liver tissue in AE patients.Part three,1)BALB/c mouse model was established with the PSCs of E.multilocularis via portal vein,and PD-1 was expressed high on CD8 Tcm at middle stage,and on CD3⁺T,CD4⁺T,CD8⁺T and Treg cells at late stage;at 12 and 24 weeks,secretion of IFN-?from PD-1⁺CD8⁺T cells was lower than PD-1^-CD8⁺T cells(14.42±1.89 vs 32.26±0.47%,4.59±0.43 vs 10.17±0.74%,P<0.001,P=0.027).2)The E.multilocularis infection was more serious in the Nude and SCID group than BALB/c group,but there was no different between the Nude and SCID group.The degree of fibrosis in the liver was higher in the BALB/c group than the Nude and SCID group(24weeks,Sirius red staining,29.35±2.56%vs 8.90±2.67%vs 9.90±1.56%,P<0.05;IHC,22.89±4.93%vs 3.84±0.98%vs 7.23±3.23%,P<0.05).3)The growth of lesion in the liver was higher in the anti-CD4⁺T cells group and anti-CD8⁺T cells group than the control group(weight 1523.00±288.50 vs 1285.00±272.60 vs 375.00±83.24mg,P=0.012,P=0.044;volume 2197.00±647.10 vs 1744.00±367.20 vs 108.80±22.63mm³,P=0.013,P=0.048),but there was no difference between the anti-CD4⁺T cells group and anti-CD8⁺T cells group.Part four,1)E.multilocularis infection was suppressed in the PD-1^-/-mouse model via portal injection(24weeks,weight 296.20±26.15 vs471.00±88.70mg,volume 221.40±29.61 vs 451.40±113.2mm³,P<0.05),with existing splenomegaly,and the degree of fibrosis of liver grew more serious(24weeks,Sirius red staining,53.56±3.77%vs 32.64±0.83%,P<0.05;IHC,27.32±6.23%vs 10.99±1.83%,P<0.05).The percentage of CD69 positive CD4⁺T cells and effective CD4⁺T cells was increased and higher in the PD-1^-/-mouse model than the BALB/c mouse model(46.44±2.06%vs 29.86±4.60%,67.12±3.96%vs 42.68±5.17%,P<0.05).The percentage of Th1 type cells increased and higher in the PD-1^-/-mouse model than the BALB/c mouse model(24weeks,IFN-?⁺CD4⁺T cells,19.26±1.90%vs 7.21±0.88%,P<0.05),but The percentage of Th2 type cells decreased and lower in the PD-1^-/-mouse model than the BALB/c mouse model(24weeks,IL-4⁺CD4⁺T cells,2.83±0.12%vs 5.53±0.92%,P<0.05).2)E.multilocularis infection was suppressed in the PD-1^-/-mouse model via subcutaneous injection(24weeks,weight 101.20±43.12 vs 393.70±123.5mg,volume82.17±28.43 vs 429.30±148.30mm³,P<0.05).3)Blockade of PD-1/PD-L1 signal pathway with antibody,there not clear suppression in PD-1^-/-mouse model,but the degree of fibrosis of liver was higher in the block group than the control group(?-PD-1,sirius red staining,37.72±2.24%vs 28.39±1.60%,P<0.05;?-PD-L1,Sirius red staining,34.15±1.70%vs 22.56±4.26%,P<0.05,IHC,25.52±3.23%vs 13.65±1.94%,P<0.05).The percentage of Th1 type cells increased and higher in the block group than the control group(24weeks,IL-2⁺CD4⁺T cells,10.75±0.74%vs 7.85±0.60%,TNF-?⁺CD4⁺T cells,22.43±1.94%vs 13.73±1.99%,P<0.05),but The percentage of Th2 type cells decreased and lower in the block group than the control group(24weeks,IL-4⁺CD4⁺Tcells,4.96±0.29%vs 7.00±0.62%,P<0.05).Conclusion:1)The expression of PD-1 on T cells is increased in AE patients and mediates the immune tolerance in T cells in the liver.2)T cells play an important role in the E.multilocularis infection,participating the course of fibrosis;the expression of PD-1 on T is increased in mouse model.3)Blockade of PD-1could suppress the growth of E.multilocularis,increase the degree of fibrosis in the liver,and return the imbalance of Th1/Th2 and Th17/Treg.