Inhibitory Molecular B7-H1 And Cell Immune Response In Chronic Hepatits B

BackgroundsIt is estimated conservatively that there are 350 million persistent carriers of HBV worldwide, 25% of whom have chronic liver disease and cirrhosis, which could progess to hepatocellular carcinoma. The annual mortality from hepatitis B infection and its sequelae is 1-2 million people worldwild. With high rate of prevalence of HBV in China, there are 150million carriers and 30million chronic hepartitis B, which represents a major public-health concern because of its propensity to progress to liver cirrhosis and hepatocellular carcinoma.The precise pathogenetic mechanisms responsible for the acute and chronic hepatitis B are only partly defined. Most studies indicate that HBV is not cytopathic for the infected hepatocyte. Studies in human and animal models provide substantial evidence that viral hepatitis is initiated by an antigen specific antiviral cellular immune response. Cell mediated immunity include CD4+T helper cells (Th) and CD8+T cells. CD4+Th orchestrate the immune response and are classified into two subsets, Th1 cells secreting IFN-γ,IL-2 and TNF-α,which mount a cell-mediated immune response defense against infection with intracellular microbes and isotype switching to IgG2a. Th2 cells secreting IL-4,IL-13,IL-5,IL-6,IL-9, IL-10 and IL-13,which are necessary for inducing the humoral response to extracellular pathogens. IL-10 is known to be a powerful inhibitor ofThl activation and to suppress CMI in mice and humans.Thl cells play an important role in anti-viral immunity by maintaining effective CTL responses, and Th2 cells dominance suppress Thl responses and may lead to immune tolerance. Thus, Thl/Th2 balances are important in the clearence of HBV in humans. During an acute HBV infection, patients exhibit a multispecitic and polyclonal CTL response and a strong Thl response. However, Chronical HBV infection is maintained by increased type-2 T helper cell response and such HBV-specific T-cell responses are generally undetectable in chronic patients.Initiation of the antigen-specific immune response requires proper interaction between the effector cell and an antigen-presenting cell. Dendritic cells are instrumental for innate and adaptive responses of the immune system. Their roles in the adaptive arm include acquisition, processing, and presentation of antigen in the context of major histocompatibility complex molecules to T cells. DC initiate adaptive immune responses in the context of cell surface MHC class II molecules and costimulatory molecules, in particular CD80 and CD86, whcih are correlated with DC functional maturation and essential for lymphocyte activation.The B7-CD28 family co-signaling molecules are crucial for the activation of T lymphocytes. These co-signaling molecules not only provide critical positive signals that stimulate T cell growth, up-regulate cytokine production and promote T cell differentiation but also contribute key negative signals that limit, terminate and/or attenuate T cell responses. In addition to the best -studied negative pathway B7-1/B7-2 - CTLA-4, recently, the new inhibitory members of the B7 and CD28 family have been identified. B7-Hl,also called programmed death ligand-1 (PD-L1), is constitutively present on monocytes, DC and could be induced on activated T cells. Ligation of B7-H1 to T cells results in the preferential production of IL-10 and inincreased T-helper-dependent synthesis of trinitrophenyl (TNP)-specific immunoglobulin G2a (IgG2a) in mice. These results suggest that ligation of B7-H1 may be responsible for promoting type 2 cytokine-biased responses.Recent studies on HBV Tg mice and chronic hepatitis B patients have suggested, however, that functional deficit of DC was an underlying cause for the T cell dysfunction. The dysfunctions included: (i) defective induction of T cell proliferative response. (ii)IL-12 secretion by DC and IL-2, IFN-y, TNF-aprodicton by T cells were decrease signcificantly. (iii) down-regulated expression of MHC-II/HLA-DR, CD80, CD86 on DC. In view of the role of B7-H1 in negatively regulating the immune response, we further investigated if B7-H1 would be involved in the functional deficit of DC.Shortage of proper immune reaction to HBV resulted from the deficit of cell mediated immune may be a major reason of chronic hepatitis B. The immune regulation aimed to enhancing the immune activity to HBV is a potential efficiency method in completely clearing HBV. Conventional protein-based vaccines are highly efficacious in preventing HBV infection;whereas in therapeutic settings with chronically infected patients, results have been disappointing. Recently a new field of immunological research and clinical application of vaccines for therapeutic purposes (vaccine therapy) has been developed for treating HBV infection.Using animal models it has been demonstrated that therapeutic vaccine immunisation strategies may overcome this problem because of their potential to induce immunity against multiple viral epitopes. DC-based vaccines mimic the effect of live attenuated viral vaccines, eliciting cell mediated immunity in addition to inducing humoral responses and has been shown to increase antigen-specific immune responses. However, efficacy may further be improved by further enhancing the ability of DC stimulating T proliferation. As blocking B7-H1 with monoclonal antibody increased DC-mediated T cells activation, we attempted to find a new method of enhancing DCvaccine efficacy by priming DC vaccine with B7-H1 monoclonal antibody.In summary, based on the negatively regulated function of B7-H1 on immune activation, we explored that the role of inhibitory molecular B7-H1 in imbalance of Th 1 /Th2, DC functional deficit, and improvement of DC vaccine efficacy.Materials and MethodsPart oneChronic hepatitis B PatientsA. Cell isolation of PBMCs.B. Expression of B7-H1, B7-DC and PD-1 on PBMCs by Flow Cytometry .C. Serum cytokine levels of IL-2, IFN-y, IL-10 were determined by using. ELISA.D. RT-PCR for B7-H1 expression in monocytes and IL-10 expression in monocytes and lymphocytes.E. Analysis of correlation between up-regulated B7-H1 expression and the higher production of IL-10.F. Analysis of correlation between up-regulated B7-H1 expression and HBeAgpresence.G. Analysis of correlation between up-regulated B7-H1 expression and serum ALT levels.Part twoHBV transgenic miceA. Spleen DC isolation.B. Expression of MHO 11, CD80, CD86, B7-H1, B7-DC on DC by Flow Cytometry .C. Analysis of T cells stimulatory capacity of DC in a mixed lymphocyte reaction.D. IL-2, IFN-y, IL-10production of T cells were determined by using ELISAE. RT-PCR for B7-H1 expression in liver tissue.F. Western blot analysis of B7-H1 expression in liver tissue.Part threeDC-based therapeutic vaccine for HBV Tg miceA. B7-H1/B7-DC effect on allostimulatory capacity of DC: groupA myeloid DC;group B myeloid DC+B7-H1;group C myeloid DC+B7-DC.B. DC vaccine preparation: loading of DC with HBsAg, MDC were cultured with 100uglIBsAglbr24h.C. Vaccine injecton: groupA, IIBsAg;groupB, HBsAg-pulsed DC;groupC HBsAg-pulsed DC with B7-H1 antibody;groupD, control group.I). Vaccine efficacy: detection of HBsAg and anti-HBsAg levels with ELISA.ResultsPart oneA Both group A and group B patients showed a significantly increased IL-10 anddecreased IL-2 and IFN-y levels levels in serum compared to healthy controls. B B7-H1-expressing CD14+ cells were significantly increased in CHB patient compared with healthy control. No significant differences in the B7-H1 and PD-1 expression by CD3+ and CD19+cells were observed between CHB patient and healthy control. No significant differences in the B7-DC expression by CD14+cells were observed between CHB patient and healthy control. C There is a significantly positively correlation between B7-H1 expression and serum IL-10 levels in group A and group B patients compared with healthy controls. D HBeAg-positive patients exhibited a higher B7-H1 expression compared withHBeAg-negative patients in group A patients.E There is a significantly negatively correlation between B7-H1 expression and serum ALT levels in group A patients.Part twoA Expression of CD8(K CD86 on DC were markedly decreased compared withcontrols. B The ability of stimulation T cell proliferation was significantly lower comparedwith controls. C The levels of IL~2^ IL-12> TNF-a in supernantant of T cells was significantlylower compared with con t ro 1 s. Part threeA Blocking of B7-H1 increased MDC-mediated T cells activation. B Administration of pulsed DCs , vaccine containing HBsAg or only completeFreund's adjuvant did not induce negative HBsAg or production of anti-HBs inany HBV-Tg mice.ConclusionsA Chronic HBV patients harbor an increased B7-H1 expression in CD 14+ monocytes compared with controls, which may be responsible for the increased serum IL-10 levels. This might be an important way by which hepatitis B virus evades an adequate immune response, leading to viral persistence and disease chronicity. B HBeAg may be responsible for the B7-H1 up-regulated expression on CD 14+monocytes. C Functional deficit of DC, partly due to decreased expression of CD80> CD86, wasan underlying cause for immune tolerance to HBV in Tg mice. D B7-H1 can negatively regulate the mature DC faction, nor was B7-DC. E HBV DC vaccine failed to elicite the humoral immune response to HBV in Tg mice, more efficient immunization strategies are needed to stimulate an immune response of sufficient quality and magnitude to achieve an immunotherapeutic antiviral effect.