Detritic Cells Pulsed With RhHSP70and HBxAg Induce Specific Anti-tumor Immune Responses In Hepatitis B Virus Related Hepatocellular Carcinoma

Dendritic cells (DCs) are powerful antigen-presenting cells （APC） invivo, which perform a critical function in the anti-tumor immunity. It is theunique one to directly activate naive T cells and then activate cytotoxic Tlymphocytes (CTL) by processing and presenting antigen to majorhistocompability complex (MHC) class I and class II molecules. However,DCs in patients with tumor are unable to induce primary immune responseto activate the CTL and the secondary immune response due to thedefection of it’s quantity and function.Primary carcinoma of the liver (mostly the hepatocellular carcinoma,so it is known as hepatocellular carcinoma, HCC, referred to "liver cancer")is highly malignant and it’s survival period is less than6months commonly.The incidence is high in our country, and accounted for54%of the world.Becorse of its insensitive to radiotherapy and chemotherapy, the maintreatment is local integrated therapy, such as operation. So, patient’soverall five year survival rate in our country is only about10%and evenlower in the middle and late stage. Recently, immunotherapy has become anew target of cancer research, especially tumor vaccine based on DCs haveachieved good results in the experimental therapy of liver cancer. Currently,domestic and international scholars have acquired a certain effect on livercancer by loading dendritic cells with tumor lysate or AFP polypeptide.However, the curative effect is limited by the lower immune response forthe single loading and the inferior immunogenicity. Therefore, it is greatsignificance to explore new loads and construct new effective DC vaccinein the clinical therapy.HBV infection is closely related with HCC, especially HBx protein(HBxAg) encoded by HBV X gene. The positive expression of HBxAg inhepatocellular carcinoma tissues is up to70%-80%, so it can be used as anideal target antigen. Furthermore, DC loaded with HBxAg will likely be anew way of the biological treatment. Besides, as a kind of important stressproteins, heat shock protein70(HSP70) family exerts the function ofadjuvant in tumor immunity. It performs as the molecule carrier to present antigen to APC surface and then activate CD8+T cells to kill tumor cells.Therefore the HSP70is also the vital significance on the development oftumor vaccine. Furthermore, HSP70posses the bioactivity to combine withpolypeptide. This research utilizes HBxAg as a target antigen and utilizesHSP70as immunologic adjuvant to prduce rhHSP-HBxAg complexs invitro. This complexes enhance the stability and antigenicity of polypeptidesand then loaded with the professional antigen presenting cells DCs toprepare rhHSP-HBxAg-DC vaccine. Then a further research on cytotoxicefficacy against HCC with positive expression of HBxAg will beunderway.Part Ⅰ Establish the loading system between rhHSP70-HBxAandDC and identify its biological functionObjective: To establish the rhHSP70-HBxAg-DC vaccine and identify itsbiological function.Methods:(1) Gather peripheral blood from healthy donors of HLA-A2+,then peripheral blood mononuclear cells (PBMCs) was separated by Ficoll,from which adherent cells were induced to DCs in vitro, observe themorphological of DCs everyday;(2) On the fifth day, DCs were loaded bydifferent concentrations of rhHSP70, HBxAg and rhHSP70-HBxAgcomplex, and then the expression of CD80, CD83, CD86, CD11c andHLA-DR were detected by flow cytometry, the secretion of IL-12wasevaluated by ELISA;(3) Separate T lymphocyte by nylon wool columnfrom the non-adherent cells, co-culture with rhHSP70-HBxAg-DC for5days in vitro, and then proliferation of T lymphocyte was detected byCCK-8kit.Results:(1) DCs appeared elongated and slender dendritic form;（2）themost suitable concentration of rhHSP-70and HBxAg are20μg/ml and5μg/ml respectively.(3) After DCs were loaded with antigen, theexpression of CD80, CD83, CD86, CD11c, HLA-DR and he secretion ofIL-12were significantly increased, the group of rhHSP70-HBxAg was themost significant.(4) rhHSP70-HBxAg-DC could stimulate the proliferationof T lymphocytes significantly.Conclusion: DCs derived from PBMCs harboring the rhHSP70and (or)HBxAg can differentiate into mature DCs, which can promote theproliferation of T lymphocytes.Part Ⅱ Cytotoxic efficacy of rhHSP70-HBxAg-DC vaccine on HCCObjective: To study the cytotoxic efficacy of rhHSP70-HBxAg-DCvaccine on hepatocellular carcinoma cells. Methods:(1) rhHSP70-HBxAg-DC co-cultured with T lymphocyte for5d,and then the cytotoxic efficacy of CTL cells in different treatmentgroups were detected by LDH release assay n vitro;(2) establish thesubcutaneously transplanted tumor model of mice with HBx-HepG2cells.30mice were divided into three groups randomly: rhHSP70-HBxAg-DCgroup, DCs group andPBS group, the volume of tumors were measuredevery3days;(3) Mice were killed and the tumors were separated on28dafter treatment. The expression of HBx, AFP, cl-2, PCNA, Fas, Bax,Caspase were detected by immunohistochemistry.Results:(1) CTL cells induced by rhHSP70-HBxAg-DC vaccine caneffectively kill HBx-HepG2cells and SMMC-7721cells, especiallyHBx-HepG2cells. But no obvious cytotoxic efficacy on K562cells (NKcells sensitive) and L02cells (hepatocyte), at the same time the cytotoxicefficacy on HBx-HepG2cells and SMMC-7721cells treated with antiMHC-class I complexes antibody decreased significantly;(2) Transplantedtumors in rhHSP-HBxAg-DC group and DCs group were inhibitedsignificantly than PBS group, and the most significantly wasrhHSP70-HBxAg-DC group;(3) Immunohistochemical staining showedthat the HBx protein and AFP is positive, and the vaccine can decrease theexpression of Bcl-2and PCNA, and increase the expression of Fas, Baxand Caspase-3in transplanted tumor tissue.Conclusion: RhHSP70-HBxAg-DC vaccine can induce a specific effect onhepatoma cells associate with HBV and this efficiency brings up a certainprospect applied to clinical therapy.