| Object:Recently,more and more reports have demonstrated that liver not only plays roles in metabolic detoxification,but also acts as an immune organ which has unique immunological characteristics.The specific structure,physiological function and microenvironment make liver possess special immune cells and effector moleculars,these also make liver is enrichment of innate immune cells(such as macrophages,natural killer,natural killer T,and ?? T cells)and the immune tolerance as the main characteristics of liver.It has reported that liver resident NK cells are different from those of peripheral and other organs,whether other kind of innate immune cells exist in liver is not known.The ?? T cells belong to innate immune system and are regard as the first line of defense infection and anti-tumor proliferation.Although its proportion is not very high,it could rapidly response to antigen,produce cytokines like interferon(IFN)-?and IL-17 to promote pathogen clearance and regulate inflammation and maintain homeostasis.Liver is rich in ?? T cells,the unique microenvironment of liver makes?? T cells have different characteristics comparing with other organs,but its relationship with liver immune tolerance and liver related diseases is not completely clear.Hepatitis B virus(HBV)persistent infection promotes the immune tolerance of liver and inhibits the immune cells(include CD8+ T and NK cells)function,even lead to cell dysfunction.In chronic HBV patients,the functions of immune cells impaire,primarily manifested as the decrease of cell number,the decline of activation,the attenuation of cytolysis ability,the increase of activating receptors and the decrease of inhibitory receptors.The dysfunction of immune cells prompts HBV escaping from the immune system surveillance and attack,but the precisely mechanism is unknown.In this study,on one hand,by comparing mouse liver,spleen,thymus and intestinal epithelial ?? T cells phenotype,we found a CXCR3~+CXCR6~+?? T cell subset which only exist in liver and had tissue-resident speciality.In HBV acute infection models,the proportion of CXCR3~+CXCR6~+?? T cells increased and secreted more IFN-?,which suggested that CXCR3~+CXCR6~+?? T cells played important roles in HBV acute infection.On the other hand,during HBV chronic infection,the transcription factor Eomes promoted the inhibitory receptors expression on CD8+ T cells,facilitated the CD8+ T cells dysfunction and HBV escaping from immune system surveillance and attack;the transcription factors GATA-2 and GATA-3 bound the HBx protein and formed a tripolymer to inhibit the expression of MICA,this could weaken the susceptibility of hepatoma cells to NK cells and facilitated the HBV positive hepatoma cells escaping from NK cells lysis.Method:1 FACS was performed to test the percentage of y8 T cells in liver,spleen,LN,BM,blood,thymus and IELs in C57BL/6J mice.2 Q-PCR and FACS was applied to confirm the expression of ?? T cells phenotype(such as chemokine receptor and adhesion marker)in liver,spleen,thymus and IELs of C57BL/6J mouse.3 FACS was performed to test the proportion,surface markers and transcription factors of CXCR3~+CXCR6~+?? T cells.4 Q-PCR was applied to confirm the ?? T cell subset in liver and thymus,FACS was performed to test the proportion,surface markers and transcription factors of CXCR3+CXCR6+y8 T cells from embryo to adult mouse.5 Establishing the CD45.1 and CD45.2 mouse parabiosis,FACS was applied to confirm the distribution of CD45.1+ and CD45.2+ CXCR3~+CXCR6~+?? T cells.6 Extracting the liver sinus endothelial cells and culture in vitro,ELISA was used to test the chemokine(CXCL9,CXCL10,CXCL11 and CXCL16)level secreted by liver sinus endothelial cells;Transwell was performed to test the chemotactic effect of liver sinus endothelial cells to CXCR3+CXCR6+y8 T cells.7 FACS was performed to test the expression of integrin P7 on liver sinus endothelial cells.8 FACS was applied to confirm the secretion of IFN-y and IL-17 and the expression of ROR?t and T-bet in CXCR3~+CXCR6~+?? T cells after PMA/ion stimulation and BFA blockade.9 FACS was performed to test the proportion of CXCR3~+CXCR6~+?? T cells in WT and GKO mice.10 FACS was used to test the level of IFN-? and IL-17 secreted by CXCR3+CXCR6+y8 T cells in HBV acute infection model.11 Flow cytometric sorting was used to sort the CXCR36+CXCR6+?? T cells and adoptive transferred these cells into TCR?-/-mouse.We established HBV acute infection model in WT,TCR?-/-and TCR?-/-mice that were adoptive transferred CXCR3~+CXCR6~+?? T cells,ELISA was used to measure the level of HBsAg,HBeAg in these three kinds of mice.12 After Establishing the HBV persistent model of mice,Flow cytometry was performed to test the percentage of CXCR3~+CXCR6~+?? T cells,the secretion of IFN-y and IL-17 and the expression of CD69,PD-1,LAG-3 and CTLA4 on CXCR3~+CXCR6~+?? T cells.13 Establishing the HBV persistent model in mice,FACS was used to test the expression of inhibitory receptors on Eomes+ CD8+ T cells,Eomes-CD8+ T cells,Eomes+ HBV-specific CD8+ T cells and Eomes-HBV-specific CD8+ T cells in spleen and liver.14 FACS was applied to test the expression of inhibitory receptors on CD8+ T cells and HBV-specific CD8+ T cells in WT and EKO mice.15 FACS was performed to test the level of TNF-a,IL-2 and IFN-y secreted by HBV-specific CD8+ T cells in WT and EKO mice.16 ELISA was used to measure the level of HBsAg,HBeAg and PCR was applied to test the HBV DNA load in serum from WT and EKO mice.Immunohistochemistry was applied to test the level of HBcAg in liver from WT and EKO mice.17 Q-PCR was performed to test the level of NFATcl,Blimpl,FoxO1 and Nfil3 and Western Blot was used to confirm the NFATcl expression in liver mononuclear cells from WT and EKO mice.18 Chromatin immunoprecipitation(ChIP)was used to test the binding of Eomes to CD 160 and LAG-3 in liver mononuclear cells.19 Q-PCR and FACS were applied to confirm the expression of MICA,MICB and ULBP1,2,3 in HepG2,HepG2-N1,HepG2-HBV and HepG2.2.15 cells.20 CFSE/7AAD was used to test the susceptibility of NKL cells to HepG2,HepG2-HBV and HepG2.2.15 cells after blocking MICA/B or NKG2D.21 Western Blot was performed to confirm the MICA expression in HepG2 cells after transfecting pEGFP-HBx,pEGFP-HBc,pEGFP-HBs and pEGFP-HBp.22 CFSE/7AAD was applied to test the susceptibility of NKL cells to HepG2-N1,HepG2-X and HepG2-C cells after blocking MICA/B or NKG2D.23 Western Blot was used to confirm the MICA expression in HepG2.2.15 cells after silencing GATA-2 or GATA-3 through transfecting GATA-2 siRNA or GATA-3 siRNA.24 In HepG2.2.15 cells,Immunoprecipitation was performed to test the binding of HBx protein to GATA-2 and GATA-3.25 Chromatin immunoprecipitation(ChIP)was used to test the binding of HBc protein to the CpG islands of MICA or MICB promter in HepG2.2.15 cells.Result:Part ? The discovery of liver-specific CXCR3~+CXCR6~+?? T cell subset1 Liver contains specific ?? T cells.By comparing the percentage of ?? T cells in bone marrow(BM),small intestine,lymph node,liver,skin,spleen and thymus,we found liver was enrichment of ?? T cells.2 CXCR3~+CXCR6~+?? T cell subset exists in liver.CXCR3 and CXCR6 were specifically expressed on liver ??T cells,the CXCR3~+CXCR6~+?? T cells expressed tissue-resident markers CD 103,CD69,CD44,CD49a and PLZF as well as transcription factor T-bet.3 CXCR3~+CXCR6~+?? T cells exist from embryo to adult mice.The liver contained V?2 and V?4 ?? T cell subsets during embryo,CXCR3~+CXCR6~+?? T cells existed from embryo to adult.The expression levels of tissue-resident markers and transcription factors in CXCR3~+CXCR6~+?? T cells were similar from embryo to adult.These suggested that CXCR3~+CXCR6~+?? T cells might develop in liver during embryo and then stay in liver.4 CXCR3~+CXCR6~+?? T cells specifially reside in liverIn the model of CD45.1 and CD45.2 parabiosis,almost all the CD45.1 +CXCR3~+CXCR6~+?? T cells existed in CD45.1 mice and nearly all the CD45.2+CXCR3~+CXCR6~+?? T cells existed in CD45.2 mice.5 Liver sinus endothelial cells promote the stay of CXCR3+CXCR6+y6 T cells in the liver.Liver sinus endothelial cells secreted high level of CXCL9,CXCL11 and CXCL16 and showed the chemotactic activity to CXCR3~+CXCR6~+?? T cells.After neutralizing the CXCL9,CXCL11 and CXCL16 pathway,the chemotactic activity of liver sinus endothelial cells to CXCR3~+CXCR6~+?? T cells was decreased.The expression of integrin ?7 on liver sinus endothelial cells promoted the CXCR3~+CXCR6~+?? T cells residing in liver.6 CXCR3~+CXCR6~+?? T cells mainly secret IFN-y.CXCR3~+CXCR6~+?? T cells secreted more IFN-y than IL-17 and expressed more RORyt than T-bet.7 The deficiency of IFN-y does not affect the percentage of CXCR3~+CXCR6~+??T cells.The percentage of CXCR3~+CXCR6~+?? T cells in WT and GKO mice had no difference.8 CXCR3~+CXCR6~+?? T cells contribute to the resistance of HBV acute infection.At 7th days after hydrodynamic injection,the CXCR3~+CXCR6~+?? T cells secreted more IFN-y.After adoptive transferred CXCR3~+CXCR6~+?? T cells into TCR?-/-mice,the level of HBsAg and HBeAg was decreased.This suggested that CXCR3~+CXCR6~+?? T cells might take part in resisting acute HBV infection.During the HBV persistent infection,the percentage of CXCR3~+CXCR6~+?? T cells decreased and the secretion of IFN-y by CXCR3~+CXCR6~+?? T cells increased.The activating marker CD69 increased and the inhibitory receptors PD-1,LAG-3 and CTLA4 were not changed on CXCR3~+CXCR6~+?? T cells.Part II The regulation of transcription factors on the expression of immune-related receptors and ligands during HBV persistent infection1 The expression of inhibitory receptors increases on T cells during HBV persistent infection.The expression of inhibitory receptors(include BTLA,CD 160,Tim-3,CTLA4,PD-1 and LAG-3)increased on CD4+ and CD8+ T cells in liver and spleen during HBV persistent infection,especially the CD8+ T cells of liver.The increase of inhibitory receptors on hepatic CD8+ T cells was more significant.2 Eomes positively correlats with the inhibitory receptors expression on CD8+T cellsThe expression of BTLA,CD 160,Tim-3,CTLA4,PD-1 and LAG-3 on Eomes+CD8+ T cells was higher than Eomes-CD8+ T cells.In addition,the expression of BTLA,CD160,Tim-3,CTLA4,PD-1 and LAG-3 on CD8+ T cells of WT mice was much higher than in EKO mice.3 The levels of inhibitory receptors on HBV-specific CD8+ T cells in WT mcie are higher than EKO mice.The levels of inhibitory receptors PD-1,Tim-3,CD 160 and LAG-3 on Eomes+HBV-specific CD8+ T cells were higher than on Eomes-HBV-specific CD8+ T cells.The expression of PD-1,Tim-3,CD 160 and LAG-3 on HBV-specific CD8+ T cells in WT mice was higher than in EKO mcie.The spice analyses showed that Eomes promoted the co-expression of inhibitory receptors on HBV-specific CD8+ T cells.4 The effector functions of HBV-specific CD8+ T cells do not differ in WT and EKO mice.The levels of IL-2,TNF-a and IFN-y secreted by HBV-specific CD8+ T cells in WT and EKO mice had no difference.5 The deficiency of Eomes promotes the clearance of HBV.The levels of HBsAg,HBeAg and HBV DNA load in serum and the HBcAg in liver from WT mice were higher than from EKO mice.This suggested that the mice increased the antiviral ability after absence of Eomes.6 Eomes binds to CD160 promoter and increases CD160 expression.The expression of Blimp-1,NFATcl and FoxO1 in WT mice was higher than EKO mice.Eomes could bind to CD 160 promoter and increased its expression demonstrated by ChIP assay.7 The expression of NKG2D ligands on HBV positive hepatoma cells is much higher than on HBV negative hepatoma cells.The HBV positive hepatoma cells HepG2.2.15 and HepG2-HBV expressed lower level of NKG2D ligands(MICA,MICB and ULBP1,2,3)compared with HBV negative hepatoma cells HepG2 and HepG2-N1.The susceptibility of HepG2-HBV and HepG2.2.15 to NKL cells was lower than HepG2 cells;after blocking the NKG2D on NKL cells or MICA/B on hepatoma cells,the susceptibility was further reduced.8 HBx and HBc downregulate the expression of MICA on hepatoma cells.After tansfected the HBx and HBc gene,the expression of MICA on hepatoma cells was reduced.The susceptibility of HepG2-HBx and HBc to NKL cells were lower compared with the control.After blocking the NKG2D on NKL cells or MICA/B on hepatoma cells,the susceptibility of NKL cells further reduced.9 GATA-2 and GATA-3 decrease the MICA expression.We predicted the transcription factors GATA-2 and GATA-3 could bind to MICA promoter.After silencing the expression of GATA-2 or GATA-3,MICA increased in HepG2.2.15 cells.10 ATA-2,GATA-3 and HBx protein form a tripolymer to inhibit the MICA expression.The ChIP assay demonstrated that GATA-2 or GATA-3 directly bound to MICA promoter.HBx protein formed a tripolymer with GATA-2 and GATA-3 and then bound to MICA promoter and inhibited the MICA expression.11 HBc protein directly binds to the CpG islands of MICA/B promoter and inhibits MICA/B expression.The ChIP assay showed that HBc protein directly bound to the CpG islands of MICA/B promoter,but HBc protein could not bind with GATA-2 and GATA-3 to form a tripolymer.Conclusions:1 By the comparison of the phenotype of ?? T cells in liver,spleen,thymus and small intestine,we found a CXCR3~+CXCR6~+?? T cell subset which only existed in the liver and these cells had tissue-resident features.In HBV acute infection model,the CXCR3~+CXCR6~+?? T cells secreted more IFN-?,adoptive transferred the CXCR3~+CXCR6~+?? T cells into TCR?-/-mouse could resist the HBV acute infection.During HBV persistent infection,CXCR3~+CXCR6~+?? T cells secreted more IFN-?than IL-17 and expressed high level of CD69.These suggested us CXCR3~+CXCR6~+?? T cells contributed to resisting HBV acute infection and might not participated in the maintance of immune tolerance,but it is necessary to further study and clarify its mechanism.2 During HBV persistent infection,we first found the transcription factor Eomes promoted the inhibitory receptors expression on CD8+ T cells,facilitated CD8+T cells dysfunction and the escape of HBV from immune system surveillance and attack;the transcription factors GATA-2 and GATA-3 bound with HBx protein and formed a tripolymer to inhibit the expression of MICA,this could weaken the susceptibility of hepatoma cells to NK cells and facilitated the HBV positive hepatoma cells escaping from NK cells lysis. |
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