| Objective: Alveolar echinococcosis(AE)is characterized by chronic and progressive hepatic damage caused by the continuous proliferation of the larval stage(metacestode)of Echinococcus multilocularis(E.multilocularis),that behaves like a so-called " parasitic liver cancer",progressively invading host tissues and organs.At present,some studies have shown that host immune status is an important factor affecting parasite infection,parasitic disease activity and prognosis of the disease.The aims for this study is to investigate the mechanism of immunosuppressive molecules TIGIT and PD-1 mediated T cells immune exhaustion in hepatic AE patients.Methods: Part one,1.Liver specimens were collected from 26 patients with AE,and the pathological changes were observed,such as the formation of "active infiltrating zone-inflammatory microenvironment," as well as inflammatory cell infiltration and liver pathology;Masson staining and immunohistochemistry were used to detect the degree of hepatic fibrosis and the activation of hepatic stellate cells in AE patients;The expression of ?-SMA,COL1A1 and COL3A1 mRNA levels was detected by the qRT-PCR;The key immune cell subsets in peripheral inflammatory microenvironment were identified by immunofluorescence technique;The levels of liver injury specific markers ALT and AST in serum were determined.2.The expression of key immunosuppressive molecules in the liver,collected from 6 patients with chronic AE,was screened by high-throughput technique;The 17 differentially expressed genes were verified by the qRT-PCR,and the correlation between the immunosuppressive molecules and the surface specific markers and the negative cell factors were analyzed.Part two,1.The liver lymphocytes were isolated by collagenase digestion,from liver specimens of 20 patients with AE,including close to the lesions and distant from lesions;The frequencies of CD4~+ T cells,CD8~+ T cells and the expression of TIGIT and PD-1 were detected by flow cytometry,and its relationship with liver lesion activity was analyzed;The localization of TIGIT and PD-1 and its ligands CD155 and PD-L1 were determined by immunofluorescence technique;The ability of TIGIT/PD-1 positive T cells and negative T cells from liver specimens of AE patients to secrete Granzyme B,perforin,IFN-? and TNF-? were detected by flow cytometry,in vitro stimulation test.2.The lymphocytes were isolated by density gradient centrifugation,from blood of AE patients and healthy volunteers;The percentages of CD4~+ T cells,CD8~+ T cells and the expression of TIGIT and PD-1 were detected by flow cytometry;The correlations between the expression of TIGIT and PD-1 on the surface of CD4~+ T cells or CD8~+ T cells and the level of serum specific antibody Em2,and liver leison activity were analyzed;The ability of TIGIT/PD-1 positive T cells and negative T cells from blood of AE patients to secrete Granzyme B,perforin,IFN-? and TNF-? and to proliferate were detected by flow cytometry,in vitro stimulation test.The proliferation of T cells was further detected by blocking TIGIT and PD-1 antibodies.Part three: 1,The size on the surface of hepatic lesions and the number of metastases in mice model with three different doses E.multilocularis infection were analyzed;Pathological sections were used to observe the pathological features of liver,the number of microscopic lesions and the type of granulomatous reaction;Sirius red staining and immunohistochemistry were used to detect the degree of hepatic fibrosis and the activation of hepatic stellate cells in AE patients;The expression of ?-SMA and COL1A1 mRNA levels was detected by the qRT-PCR;The liver microscopic lesion size and absolute number of mononuclear cells were measured.2,The absolute lymphocyte count,the T lymphocyte phenotype(CD69,CD44 and CD62L)and the percentages of T cell subsets,including T1-,T2-,T17-and Treg-type T cells isolated liver mononuclear cells,were measured by flow cytometry.The mRNA levels of T cell subsets related cytokines in liver tissue were detected by the qRT-PCR.3.The expression of TIGIT and PD-1 on the surface of CD4~+ T cells and CD8~+ T cells from liver and spleen of mice model were analyzed by flow cytometry;The ability of TIGIT/PD-1 positive T cells and negative T cells to secrete Granzyme B,CD107 a,IFN-? and TNF-? were detected by flow cytometry,in vitro stimulation test.Results: Part one,1.The inflammatory microenvironment was formed around the liver lesions in patients with chronic AE,which was mainly composed of CD8~+ T lymphocytes infiltration,accompanied by infiltration of CD4~+ T lymphocytes,macrophages and fibroblasts.2.High throughput screening of gene chip showed that the chemokines(CCL3,CCL8,CCL17,CCL18,CCL19,CCL21,CCL26),lymphocyte surface molecules(CD69,CD44,CD1 E,CD8B,CD27),immunosuppressive molecules(CTLA4,CRTAM,BTLA,TIGIT)and inflammatory cytokines(IL-10,IFN-?,IDO1)were up-regulated in liver tissue close liver lesion in AE patients,of which TIGIT,IL-10 and IFN-? were up-regulated 2.07-fold,3.72-fold and 3.19-fold,respectively.In addition,the positive correlations were found between the levels of TIGIT and PD-1 mRNA and CD4 and CD8;The level of TIGIT mRNA was positive correlated with PD-1.Part two,1.The levels of TIGIT and PD-1 expression on the surface of CD4~+ T cells or CD8~+ T cells were up-regulated alone or synergistically in liver specimens of AE patients,resulting in a decreased ability of local CD4~+ T cells and CD8~+ T cells in liver to secrete Granzyme B(CD8~+PD-1+ vs CD8~+PD-1-: 8.00±3.03% vs 32.0±8.32%,P<0.05),IFN-?(CD4~+TIGIT+ vs CD4~+TIGIT-: 8.18±2.65% vs 17.8±3.95%,P<0.05)and TNF-?(CD8~+TIGIT+ vs CD8~+TIGIT-: 9.67±2.56% vs 18.9±3.92%,P<0.001;CD8~+PD-1+ vs CD8~+PD-1-: 7.22±2.71% vs 24.7±4.48%,P<0.01).2.Hepatocytes around liver lesions in AE patients for CD4~+ T cells or CD8~+ T cells surface TIGIT provided ligand CD155,and macrophages provided ligand PD-L1.3.The percentage and absolute number of CD8~+ T cells significantly increased in blood of AE patients;And the levels of TIGIT and PD-1 expression on the surface of CD4~+ T cells or CD8~+ T cells were up-regulated alone or synergistically in blood of AE patients,resulting in a decreased ability of liver local CD4~+ T cells and CD8~+ T cells to secrete Granzyme B,perforin,IFN-? and TNF-?;in addition,the proliferation ability of TIGIT positive cells in CD8~+ T cells decreased significantly(CD8~+TIGIT+ vs CD8~+TIGIT-: 38.7% vs 67.7%,P<0.05).The percentage of PD-1 positive CD4~+ T or CD8~+ T cells increased with the increase of serum anti-Em2 antibody levels in patients with AE(CD4~+PD-1+ T cellss,Em2,+ vs ++ vs +++: 3.41±0.70% vs 6.91±1.00% vs 6.91±1.00%,P<0.05).Through TIGIT antibody blocking,it can promote the proliferation of CD4~+ T cells and CD8~+ T cells.4.According to the PET/CT imaging group,the percentage or absolute number of CD8~+TIGIT+ T cells significantly increased in liver and blood from the highly active AE patients(liver,high vs low: TIGIT,31.7±9.09% vs 26.4±11.0%;PD-1,19.8±8.57% vs 12.1±2.08%,P>0.05.blood,high vs low: 51.6±14.7% vs 38.2±15.1%,P<0.05;PD-1,22.1±11.7% vs 15.9±10.0%,P>0.05).Part three,1.The mice model inoculated via the hepatic portal vein with different dose E.multilocularis protoscolex were successfully established.mice with low and medium dose infection were able to mount the predominance of T1(CD4~+IFN-?+ and CD8~+IFN-?+)and T17-type T cell subsets in liver,leading to parasite clearance in the early stage,and mice with high dose infection might form " liver inflammatory microenvironment ",which was mainly composed of CD8~+ T lymphocytes infiltration,and the predominance of T2-,T17-and Treg-type T cell subsets in liver,leading to chronic infection in liver.2.The levels of TIGIT and PD-1 expression on the surface of CD4~+ T cells or CD8~+ T cells were up-regulated alone or synergistically in liver of mice with high dose in the middle and late stage(24 weeks,CD8~+TIGIT+ T cells,con vs low vs medium vs high dose group: 8.91±2.29% vs 12.3±1.45% vs 8.90±0.99% vs 15.5±2.15%,P<0.05),resulting in a decreased ability of liver local CD4~+ T cells and CD8~+ T cells to secrete Granzyme B,CD107 a,IFN-? and TNF-?.3.The levels of TIGIT and PD-1 expression on the surface of CD4~+ T cells were up-regulated alone or synergistically in spleen of mice with different dose in the middle and late stage(24 weeks,CD4~+TIGIT+ T cells,con vs low vs medium vs high dose group: 15.7±2.54% vs 28.8±2.10% vs 27.6±3.46% vs 28.0±2.82%,P<0.05;CD4~+PD-1+ T cells,con vs low vs medium vs high dose group: 28.2±4.20% vs 42.6±2.59% vs 45.6±5.25% vs 39.7±2.41%,P<0.05),resulting in a decreased ability of spleen CD4~+ T cells to secrete IFN-? and TNF-?;Moreover,the ability of spleen CD8~+ T cells to secrete Granzyme B,CD107 a,IFN-? and TNF-? was decreased.Conclusion: 1.The inflammatory microenvironment is formed around the liver lesions in patients with chronic AE,which is mainly composed of CD8~+ T lymphocytes infiltration,and around where hepatocytes provide ligand CD155,and macrophages provide ligand PD-L1,and the inhibitory signal transduction may be mediated by CD155-TIGIT and PD-L1-PD-1 binding patterns,resulting in the decrease of liver local T cells function,and promoting liver local T cells exhaustion.2.According to the PET/CT imaging group,the percentage or absolute number of CD8~+TIGIT+ T cells significantly increased in liver and blood from the highly active AE patients,resulting in decreased function of T cells and active or invasive growth of E.multilocularis.Through TIGIT antibody blocking,it can promote the proliferation of CD4~+ T cells and CD8~+ T cells.3.The mice model inoculated via the hepatic portal vein with different dose E.multilocularis protoscolex were successfully established;The immune imbalance of T1-,T2-,T17-and Treg-type T cell subsets,and the up-regulated expression of TIGIT and PD-1 on the surface CD4~+ T cells and CD8~+ T cells in liver or spleen inhibit the function of T cells from mice with high dose infection,leading to the chronic parasitism of E.multilocularis in the liver and the pathological liver injury and fibrosis. |
PDF Full Text Request