Dendritic Cells Pulsed With Recombinant Human HSP70 And Freeze-thaw Cell Lysats Derived From HepG2 Cells Induce Specific Anti-tumor Immune Responses

ObjectiveDendritic cells(DCs) are the most powerful antigen presenting cells,which are able to induce the cellular immune response through antigen presentations.DCs can activate priming naive T lymphocytes and have ability to induce immune response against malignant cells.DC-based anti-tumor vaccines have made exciting progress in experimental study and clinical research in recent years,which have shown a wide range of applications,but there is still a major problem in course of the preparation of cancer vaccines that the antigen immunogenicity is so weak as not to present antigen to activate cytotoxict lymphocytes(CTLs).In order to improve its immunogenicity,an adjuvant must be added.HSP70 is an immune adjuvant,promotes immature DCs into mature DCs,stimulates the secretion of cytokines and enhances immune function.In our study,the phenotypic and morphological changes of DCs were observed before and after they were pulsed with recombinant human heat shock protein 70(rhHSP70) and freeze-thaw cell lysats derived from HepG2 cells.As the same time,the outcome of induced specific CTL-mediated cytotoxicity were obtained,too.In our study,we wanted to examine if CTLs induced by DCs pulsed with rhHSP70 and freeze-thaw cell lysats derived from HepG2 cells can specifically killed HCC cells in vitro or not,and also we analysed the mechanism which the potent anti-tumor immunity is induced by DC-based vaccines after rhHSP70 is added.Our study can provide a experimental basis and theoretical foundation for designing DC-based high efficient vaccines against HCC. MethodsDCs were derived from peripheral blood mononuclear cells of healthy donors and then were stimulated in vitro with granulocyte-macrophage colony-stimulating factor(GM-CSF) and interleukin-4(IL-4).DCs were loaded with freeze-thaw lysate derived from HepG2 cells,at the same time rhHSP70 as an immune adjuvant was added.The different groups of DCs induced different proliferation of tumor specific cytotoxic T cells(CTL).DCs were divided into four groups:①group A:DCs+ the freeze-thaw lysate(Ag);②group B:DCs+rhHSP70;③group C: DCs+Ag+rhHSP70;④group D(the control group):solitary DCs.The different group of DCs activated CTLs respectively.DCs phenotypes(CD80,CD83,CD86) were observed by FCM.Morphological characteristics of DCs were viewed by the inverted microscope and the scanning electron microscope(SEM).The anti-tumor effect activated by DC-based vaccines and the proliferation of lymphocytes were measured by MTT assay.The HepG2 cells apoptosis were viewed by fluorescence staining.ResultsThere was a significant difference among the control group and other three ones in the phenotypes of DCs(P＜0.05).The positive expression rate of CD80:80.2±2.5%in group A,79.6±2.6%in group B,81.6±2.3%in group C and 21.5±1.4%in group D.The positive expression rate of CD83:71.3±2.9%in groupA,68.3±2.4%in group B, 73.4±2.1%in group C and 33.4±2.4%in group D.The positive expression rate of CD86:74.2±3.6%in group A,75.3±4.8%in group B,82.5±5.0%in group C and 48.2±2.8%in group D.The DCs pulsed with rhHSP70,freeze-thaw lysate and combination had more protrusions than that of the control group in the light microscopy and SEM,which shows they were in maturate state.The modified groups of DCs had maturate morphological characteristics,but the control group of DCs had immaturate morphological characteristics.The result revealed that the different groups of DCs had different abilities of stimulating lymphocytes proliferation.The ability of proliferating is described by proliferation index(PI,PI= OD experiment/OD control).The results were as followed: 2.10±0.05 in group A,2.06±0.05 in group B,2.60±0.06 in group C and 1.20±0.05 in group D.PI of group C was at the highest-level among four groups,Group A followed by.There was a significant difference among the control group and other two experimental groups(groupA and group B)(P＜0.05).There was not a significant difference between that of groupA and group B(P＞0.05).Cytotoxicity against HepG2 induced by different group of DCs was not same. Cytotoxicity=(1-OD experiment/OD control)×100%.The results of cytotoxicity in four groups at three ratioes of effector cells / target cells(E/T)(10:1,20:1 and 50:1) were as followed respectively:40.27±4.89%,47.13±8.05%and 51.64±6.73%in group A;13.58±4.77%,12.13±4.26%and15.57±5.55%in group B;61.09±7.89%, 64.71±3.90%and 74.59±3.34%in group C;11.94±4.11%,13.90±4.11%and 13.52±4.61%in group D.The cytotoxicity in group C was the highest at the three ratioes of E/T,which was significant compared with the three others(P＜0.05)and especially much higher than that in group D(P＜0.05).Yhe result also reaveled cytotoxicity in group A followed by.There was no significant difference beteen the cytotoxicity in group B and that in group D(P＞0.05).ConclusionDCs pulsed with freeze-thaw cell lysats derived from HepG2 cells can enhance the growth expansion of lymphocytes,present effectively tumor antigen to stimulate generation of specific CTLs,which were very effective in activating specific T-cell responses against HepG2 cells in vitro.The reason that anti-tumor immunity induced by DC-based vaccines was improved may be related to the maturation of the DCs promoted by rhHSP70.