Discrepancy And Reversibility Of INKT Cell Defects During Chronic Hepatitis B And Hepatocellular Carcinoma

iNKT cells possess both T and NK cells surface markers and functional characteristics,thus serve as a bridge between innate and adaptive immunity.The enrichment of iNKT cells in liver suggests they may play roles in hepatic disease.TCR of iNKT cells recognize lipid antigens presented by CD1 d.?-GalCer is the strongest agonist for iNKT cells.HBV is a hepadnavirus DNA virus.In China,about 7.18% of total population exhibits sustained hepatitis B surface antigen positive.Chronic HBV infection results in a series changes in the liver,which is believed to cause chronic hepatitis,cirrhosis and hepatocellular carcinoma.iNKT cells exhibit strong antiviral and antitumor effects in mouse models.However,a randomized placebo controlled phase ?/? trial indicates that ?-GalCer administration failed to promote sustained anti-viral activity in CHB patients.The possible explanation would be that little is known about characteristics of iNKT cells during chronic HBV infection,which limits the establishment of iNKT cell-based immunotherapeutic methods.In this study,we tried to determine iNKT cell number and function changes in the samples of chronic HBV infected patients.To analyze possible reasons for the failure of the clinical trial.To explore possible ways to recover iNKT cells number and function based on their specific agonist ?-GalCer.This study would provide experimental basis for human iNKT cell antiviral and antitumor applications.1.The changes of iNKT cells ratio during chronic HBV infectionWe collected peripheral blood samples from 242 healthy donors and 183 chronic HBV infected patients.The chronic HBV infected patients were classified into immune tolerance individuals,CHB patients,inactive carriers and HCC patients.iNKT cells were marked with CD3 antibody and PBS57/CD1 d tetramer and detected by flow cytometry,to observe the changes of circulating iNKT cell frequency during different phases of chronic HBV infection.Biopsy specimens of 4 HBV negative hepatic hemangioma patients and 6 HCC patients were used for the hepatic iNKT cells frequency.Results showed that iNKT cells ratio exhibited a skewed distribution among humans.Compared to healthy donors,circulating iNKT cells ratio was reduced in chronic HBV infected patients.The reduction was related to the progress of chronic HBV infection,i.e.,no reduction in iNKT cells was observed for the patients at immune tolerance phase,a significant reduaction for the inactive carriers,while CHB and HCC patients had the fewest iNKT cells.The decline of iNKT cells was also related to liver injury degree and HBV DNA level,the patients with severer liver injury and high HBV DNA level tended to have fewer iNKT cells.In the meantime,hepatic iNKT cells ratio among HCC patients was decreased.The higher expression of CD69,Fas and Fas L suggested activation-induced cell death would be a cause for iNKT cells reduction in the CHB patients.2.Functional changes of iNKT cells in chronic hepatits B and hepatocellular carcinoma patientsiNKT cells from healthy donors could be expanded and produced large amounts of Th1 bias cytokines(IFN-?)by ?-GalCer stimulation.However,an ?-GalCer administration phase I/II trial fail to show viral clearance activity in CHB patients.We found that both circulating and hepatic iNKT cells from CHB and HCC patients exhibited hyporesponsiveness to ?-GalCer stimulation.The proliferative ability and IFN-? production were reduced in CHB and HCC patients,which was related to liver injury.The hyporesponsiveness could be attributed to the ratio change of subsets of iNKT cells in the patients,as IFN-? secreted CD4-CD8-iNKT cell subset was dimished.The ratio reduction and functional loss of iNKT cells in CHB and HCC patient suggested their antiviral and antitumor ability was decreased with disease progression and liver injury.3.iNKT cell related potential antiviral and antitumor applicationsWe proved that iNKT cells expanded from healthy donors exhibited cytotoxicity to CD1d-expressing hepatoma cells in vitro.Hepatic CD1 d level were correlated of HBsAg expression when compared by immunofluorescence,which suggested HBV infected tissues could be a target for iNKT cells.Exogenous IL-2 and IL-15 partially restored iNKT cells responsiveness to ?-GalCer among CHB patients.Although the IFN-? production of iNKT cells in HCC patients was restord by these cytokines,the restoration was not observed for the iNKT cell proliferation.These results suggested the usage of both ?-GalCer and the cytokines might break the iNKT cells hyporesponsiveness status in CHB patients.On the other hand,adoptive transfer of iNKT cells in vitro expanded from healthy individuals might be a potential therapy for HCC patients.Our observations help to understand the defects of iNKT cells in the patients and develop corresponding immune interference.Conclusions: 1.This research revealed reduction of circulating and hepatic iNKT cells ratio among chronic HBV infected patients,which was related to disease progression and liver injury.2.We found that iNKT cells from CHB and HCC patients have intrinsically defects,which determined their hyporesponsiveness to ?-GalCer stimulation.These findings explained the uselessness of ?-GalCer administration in CHB patients.3.The possibility to recover iNKT cells from the defects was discussed.Exogenous IL-2 and IL-15 could partially recover ?-GalCer induced iNKT cells proliferation and IFN-? production in CHB patients.The cytokines were able to restore IFN-? secretion for iNKT cells in HCC patients,but no restoration was observed for the iNKT cell expansion.Our findings provided a strategy for establishing iNKT cell-based immune intervention.4.The association between hepatic CD1 d level and HBV infection was observed in liver tissues of HCC patients.iNKT cells from healthy donors showed cytotoxicity to CD1d-expressing hepatoma cells.As the expansion defects was not able to be resored in HCC patients,suggesting adoptive transferring iNKT cells in vitro expanded from healthy donors might be a potential therapy for HBV infected HCC.Original Pionts in this study 1.We found that the failure of clinial trials for ?-GalCer administration in CHB patients might be attributed to the intrinsic defects of iNKT cells.Recovery of the iNKT cells from the defects was observed by exogenous cytokines.2.The association between hepatic HBsAg and CD1 d expression were found,which provided a potential target for iNKT cell related therapy during chronic HBV infection.