The Function And Mechanism Analysis Of CD4~+CD25~+ Regulatory T Cells In Poor/non-responsiveness To Hepatitis B Surface Antigen Vaccine

The hepatitis B virus(HBV) is a noncytopathic DNA virus that causes both acute and chronic liver disease.More than 5%of the world population,35 million people,are currently infected with HBV,and＞250,000 people die each year from HBV-related liver cirrhosis and hepatocellular carcinoma.The development of antiviral and immunotherapies for HBV-infected patients therefore remains an important priority.China is a big country with an increased infection of HBV,many people are suffering the puzzle of this virus infection.So for the sake of effectively control virus spreading abroad, precaution seems very important now.For nearly two decades,protective vaccines that against hepatitis B virus infection have been administered routinely and the antibodies to the hepatitis B surface antigen(HBsAg) they elicit are clearly effective in preventing infections with HBV.However,bases statistical investigation after standard immunization there is still about 5%～10%of healthy vaccine recipients fail to produce protective levels of antibodies (defined as an antibody level＜10 IU/L)to the hepatitis B vaccine,show poor or non-responsiveness.And these individuals remain at risk of infection with HBV.This phenomenon has been observed in all surveys of vaccine effectiveness,irrespective of the HBsAg vaccine employed,and its cause remains largely unknown.Recently,some idears had been tabled.They thought that weather it is because antigen presenting cells(APC) of NR are defective in the uptake of HBsAg and that they are unable to present this antigen adequately,or B7-costimulation molecule is deficient expressed in nonresponders and lack of HBsAg-specific T cell clones in their peripheral blood.But these series of hypothesis had been soonly denied by subsequent experiments.Immunological tolerance has developed to allow organisms to differentiate between self and nonself.T cell tolerance is achieved primarily through the elimination of potentially autoreactive clones in the thymus through a mechanism known as negative selection.The clones that escape central tolerance in the thymus are rendered anergic in the periphery upon encounter with an antigen under suboptimal conditions.In spite of these two mechanisms for maintaining tolerance,autoreactive T cells can be readily detected in normal individuals. Recently,a CD4~+ CD25~+ regulatory T(Treg) cell population has been identified and shown to actively suppress immune responses.These Treg cells,characterized by the expression of the cell surface markers CD4 and CD25,inhibit the activation of autoreactive T cells in an antigen-specific,cell-contact dependent manner.In these decades,CD4~+ CD25~+ regulatory T cells have been shown to play a critical role in immunologic self-tolerance as well as antitumor immune responses and transplantation. And have an immunosuppressive effect on the activation and proliferation of other CD4~+ and CD8~+ T cells.Recently it is reported that chronic HBV patients who are characterized by a weak immune response to HBV harbor an increased percentage of Treg in peripheral blood compared with controls.The presence of HBV-specific Treg could contribute to an inadequate immune response against the virus,leading to chronic infection.And Hyung w Lim et al indicated that in human tonsils Tregs can migrate to GCs and regulate the helper activity of the T cells in GCs(GC-Th cells),and found this subset of T cell in human tonsils that displays potent suppressive activities toward GC-Th cell-dependent B cell responses.Based above background,we make a hypothesis that whether up regulation of Treg is involved in poor or non-HBsAb production population after hepatitis B vaccine and weather it is physiologically relevant to Th cell-dependent B cell antibody responses.Becouse these years it is confirm that CD4~+ CD25~+ double positive T cells are not really regulatory T cells.CD25 is a marker of T cells' activation,so CD4~+ CD25~+ double positive T cells are include partly activited CD4~+ T cells.And now many studies have reported that, FOXP3,a forkhead/winged helix transcription factor,is overexpressed in CD4~+ CD25~+ Treg, plays a key role in the development and function of CD4~+ CD25~+ Treg to some extent.So now FOXP3 is generally accepted a especially marker for Treg and define that CD4~+ CD25~+ FOXP3~+ T cells are really regulatory T cells.CD4~+ CD25~+ FOXP3~+ regulatory T cells(Treg) have emerged as a unique population of suppressor T cells that maintain peripheral immune tolerance.Although the immunoregulatory ability of Treg is no longer contested,where and how this population of CD4~+ T cells is generated and developed still remains largely unknown.The major debate centers on whether Treg are generated only in the thymus from a defined lineage and/or whether these cells represent a stage that different types of CD4~+ T cells can acquire. Although most analyses emphasize the thymus as the sole incubator for Treg,there are some evidence suggests that Treg may also be induced in the periphery with antigen.Treg's development,peripheral maintenance,and suppression function are dependent on Ag specificity.Battaglia M et al recently reported that rapamycin allows in vitro expansion of murine CD4~+ CD25~+ FoxP3~+ Tregs,which preserve their suppressive function and these rapamycin-expanded Tregs can suppress the proliferation of both syngeneic and allogeneic CD4~+ and CD8~+ T cells.Emma L et al identify that Ag-specific Treg from NOD mice were efficiently expanded in vitro with recombinant islet peptide,the expanded Ag-specific Treg expressed prototype surface marker and cytokines and were still capable of bystander suppression.These results imply that effective clinical therapy will benefit from the ability to expand relevant Ag-specific Treg.Except these studies,now several experimental models support that naive Foxp3~- CD4~+ CD25~- T cells can be converted in the periphery into Foxp3~+ Treg cells with antigen,the CD4~+ CD25~+ Treg cells developing de novo in the periphery were also capable of suppression function both in vitro and in vivo.For example,Mario delgado et al reported that the vasoactive intestinal peptide,an immunosuppressive neuropeptide,could induce CD4~+ CD25~+ FOXP3~+ functional Treg cells in vivo from the CD4~+ CD25~- FOXP3~- T cells.And recently,Polanczyk et al reported that estrogen promotes tolerance by expanding the CD4~+ CD25~+ Foxp3~+ -expressing regulatory compartment from CD4~+ CD25~- T cells.So it is affirmed that the Treg cells have antigen specificity and they could be induced by antigen from the CD4~+ CD25~- FOXP3~- T cells.We all know that asthma characterized by chronic airway inflammantion,invove a number of different inflammatory cells,inflammatory mediators,and cytokines.Recently many researches indicated that the activation of effector CD4~+ T cells,especially Th2 cells which could produce many inflammatory mediators and cytokines played a key role in the pathogenesis of asthma,and CD4~+ CD25~+ Treg probably could inhibit the development of asthma through inhibiting the activation of Th2 cells.So in this paper we will explore the phenomenon of poor/non-response to hepatitis B surface antigen vaccine by analyzing:the frenqeucy of CD4~+ CD25~+ Treg in poor or non-responders by Flow cytometric analysis and detecting FOXP3 mRNA expression with RT-PCR and then decision the relationship between Treg and poor/ non-responsiveness; detecting PBMC proliferation after stimulation with HBsAg and to observe weather can induce a population of antigen specific CD4~+ CD25~+ Treg from poor or non-responsiders and to define the antigen specificifity of Treg and their suppression function;detecting weather can induce a new subset of antigen specific CD4~+ CD25~+ Tregs from CD4~+ CD25~- T cells in poor or non responsiders after stimulated with HBsAg and detecting its FOXP3 mRNA expression after stimulated;because IL-4 play a important role in Th2 cells' polerization and it has been mainly secreted by Th2 cells for helping B cells antibody producting,so we will detete the production of intracellular cytokine-IL-4 by flow cytometric analysis after depleted and didn't depleted of Treg from poor or non-responsiders and then define weather these Tregs can inhibit the secreting of IL-4,therefore inhibit the Th2-depended B cells antibody production.The results showed that there is a high percentige of CD4~+ CD25~+ FOXP3~+ Treg in peripheral blood of poor/non responders compared with high respondrs;and this subset of Treg has HBsAg specificity,they can inhibit the proliferation of PBMC in poor/non responders;and we also found that we can induce a new subsut of HBsAg specific CD4~+ CD25~+ FOXP3~+ Treg from CD4~+ CD25~- T cells in poor/non responders;and in Treg depleting experiment,we found that Treg can inhibit the secreting of IL-4.by Th2 cells in poor/non responders,so implicated that Treg may potentially inhibit the Th2-depended B cells antibody response.