Coinfection Of RAAV/AFP Enhances The Immunostimulatory Effect Of Human Peripheral Blood Monocyte Dendritic Cells

ObjectiveDendritic cell(DC) is the strongest antigen presenting cell(APC).It can present antigen to T lymphocytes in vivo and in vitro, stimulate T cells reaction and induce cytotoxic T lymphocyte(CTL) reaction. Adeno-associated virus (AAV) vectors are gaining popularity for the treatment of genetic disease. Not only can they transfer genes into cells with great efficiency, they also have the surprising ability to promote long-term gene expression in animals, which typically increases with time after vector administration.The AFP is to be synthesized by the embryo yolk and liver of embryo.After be born, the level of AFP in blood descends quickly.Arriving to became adult,AFP was almost hard examinated.The AFP in 2/3 of the patient of HCC's serum had gone up.The articles had reported the way,which the AFP can repress the host immune system in the body and evade the immunity surveillance to promote the tumor indirectly by tumor neucrosis factor family;In addition, the experiment outside the body also had confirmed,the AFP can promote the growth of parts of tumor cells directly.This study was designed the immunostimulatory effect on human peripheral blood monocyte derived dendritic cells coinfected with recombinant adeno-associated virus expressing alpha fetoprotein antigen(rAAV/AFP) and provide technologymethods for clinical tumor-therapy.Methods1.Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated from buffy coats by gradient density centrifugation in Ficoll-Hypaque as previously described. DCs were prepared according to the method of Liu Y et al, with the following modifications: On Day 0, five million PBMCs per well were seeded into six-well culture plates serum-free AIM-V medium,and one million PBMCs per well were seeded into twenty fourwith-well culture plates serum-free AIM-V medium. The PBMCs were incubated at 37°C and 5% CO2 for 4 hr and the non-adherent cells were harvested by gentle pipetting; the remaining adherent monocytic cells were further cultured in AIM-V medium containing 800 U/ml of recombinant human granulocyte/macrophage colony-stimulating factor(GM-CSF).Newly isolated DCs precursor were coinfected with rAAV/AFP.After 12 hr, rAAV/AFP were gently washed off. The culture medium was removed with care not to disturb the loosely adherent cells, and 2.5 ml per well or lml per well of new AIM-V medium 800 U/ml of recombinant human GM-CSF was added and the cells were cultured at 37°C and 5% CO2.On Day 3, 2.5 ml of fresh AIM-V medium containing 800 U/ml of GM-CSF and lOOOU/ml of interleukin 4(IL-4) was added to the culture. On Day 5, DCs in suspension were gently remained and the culture medium was changed. The culture medium still contained GM-CSF and IL-4. On Day 6, tumor neucrosis factor a(TNF-a) was added to the culture. On Day 7, DCs was matured.2.DCs were observed and compared by invered phase congtrast microscope and photographed every day.3.DCs were collected in the culture process from 3rd day to 11th day.Cells in suspension was equivalent mixtured with 1% trypan blue solution.Number of viable cells was respectively counted by invered phase congtrast microscope within 3minutes.The percent of viable DCs was understood by trypan blue exclusion every24 hours.4.DCS were immunostained with anti-CD80, anti-CD86, anti-CD83, anti-HLA-DR mAbs of fluorescein isothiocynate(FITC) or phycoerythrin(PE) and analyzed by fluorescence-activated cell sorting(FACS).5.After DCs were coinfected with rAAV/AFP in three days,we collected cells and appended anti-AFP mAbs.Under the aphotic condition of 4°C, DCs were hatched for 40 mins.The PBS washed cells twice.Rabbit anti-mouse antibody of FITC was appended into cells in suspension and cells were hatched for 40 mins. The PBS washed cells twice.The infected efficiency of rAAV/AFP were detected by means of FACS after transfection.6.The non-adherent cells were kept for incubation in RPMI1640 medium with 5% human AB serum in the presence of 200U/ml IL-2. On Day 4,complete medium was changed.7.After the maturity of DCs,the non-adherent cells were relocated from the teeth outwards nylon hair pillar and drilled through nylon hair pillar on condition of 37°C and 5% CO2 for 30 mins.The non-adherent cells were qua T cells. DCs were suspended by RPMI-1640 total culture solution,and added into final concentration25 ug/mL of mitomycin C(MMC),after incubated 30 min at 37°C,washed 3 times with RPMI-1640 culture solution,adjusted cell concentration to 4xlO5/mL as irritating cell.Then DCs and T cells were mixtured according to 1:15,which was ratio of cells number. After 3 days,T cells were immunostained with anti- CD3- PE, anti- CD8- PE,anti- CD4- FITC mAbs and analyzed by FACS.8.Meanwhile,the stimulating T cells reaction were measured by method of3H-thymidine uptake after four days of DC and T cells co-culture. 9.Statistical significance of counting differences between the two groups DCs wasdetermined by applying Nonparametric test of 2 related samples. Statistical significance of SI differences between the two groups DCs was determined by applying Independent-samples t test.We defined statistical significance as p < 0.05.Result1 .Large amount of mature DC could be obtained from monocytes by culture within 7 days.DC showed dramatic changes in cell morphology.Many dendritic projections appeared at the adherent cells gradually observed by light microscope.AU kinds of DC composed of cluster by the projections.2.DCs were counted by trypan blue exclusion every 24 hours from 3rd day to 11th day. No significant change in the percent of viable DCs were detected after transfection by applying Nonparametric test of 2 related samples(p>0.05).3.FACS analysed superficial markers of two groups DCs:Markers percentage of rAAV/AFP-DC group were CD80/28.76,CD86/92.48,CD83/4.46,CD40/95.23, CDla/53.19,HLA-DR/70.37. Markers percentage of N-rAAV/AFP-DC group were CD80/30.05,CD86/88.33,CD83/7.83,CD40/88.93,CDla/42.46,HLA-DR/77.06.Two groups together highly expressed CD86, CD86 and HLA-DR.4.After DCs precursor were coinfected with rAAV/AFP, 77.7% of them expressed AFP protein.5.We compared T cells markers between the two groups, no significant difference in the CD3,CD4,CD8 and CD4/CD8 were detected after transfection by by applying Independent-samples t test(p>0.05).6.After the stimulating T cells reaction were measured by method of 3H-thymidine uptake,we compared SI of T cells between the two groups, no significant difference in the SI were detected after transfection by by applying Independent-samples t test(p>0.05).Conclusionl.In vitro,amounts of DCs could be harvested in despite of coinfection.lt is notobvious different in respect of growth process and the appearance characteristic bylight microscope. 2.whether the mature DCs were coinfected by rAAV/ AFP,growth restrain andcytotoxic reaction wasn't found. 3.No significant change in the percent of viable DCs and the surface markers onmature DCs were detected after transfection. 4.After being coinfected with rAAV/AFP,about 77.7% muture DCs expressed AFPprotein. 5.It were not differences about ability of making T cells activation between the twogroups DCs. 6.Transfected DCs still have strong potential to stimulate the proliferation of T cells.AFP gene,which was carried by recombinant adeno-associated virus,could be transferred into DCs with high efficiency. The function of mature DCs was not affected significantly by the transfection of AFP.The results of this research provide the basic materials for the DCs based vaccine against HCC.