Study Of Immunotherapy On HCC With IL-2 Engineered Human Dendritic Cells

Objective1 studied the expressing of membrane molecules of dendritic cells in peripheralblood of HCC patients. 2 Compared the differences of surface phenotype and immuno-status ofdendritic cells of HCC patients with that of the healthy persons. 3 Established the IL-2 gene recombinant adenovirus vector. 4 To analyze the variations of the surface phenotype and immuno-status ofdendritic cells that they had been IL-2 gene modified. 5 To explore specific CTL activity induced by IL-2 gene engineered dendriticcells loaded with tumor antigen.Methods 1 Generation of dendritic cell from peripheral blood of healthy persons andHCC patients.We used Ficoll Hypaque density gradient centrifugation to isolate the mononuclear cells in the peripheral blood. After 2 hours incubation, the nonadherent PBMCs were removed by washing with medicum and then fresh RPMI-1640 culture medium was added and supplemented with GM-CSF 500 u /ml IL-4 500 u /ml. Culture medium was refreshed every other day.Supplemented TNF- a 200 u/ml into culture medium at the sixth day to promote immature dendritic cells becoming mature stage. Mature DCs were taken pictures using focus scanning microscope and electron microscope. Mature DCs were also analyzed by flow cytometry with labeled monoclonal antibodies. The stimulatory effect of mature DCs on T cells was determined by T cells proliferation assay using the means of 3H-TdR incorporation. Made comparison between the healthy group and patients group. 2 Construct adenovirus vector containing human IL-2 gene and transfected 293packaging cells.Human IL-2 gene was generated by PCR amplification from Pc DNA3 and linked with enzyme digestion sequence xbaI and kpnI. After digestion with xbaI and kpnI the plasmid pshuttle was recombinant with IL-2 cDNA. The pshuttle/IL-2 recombinant plasmid transduced E coli JM 109. Positive clone had been selected. The pshuttle/IL-2 plasmid had been digested again by Pl-scel and I-cel enzyme digestion sequence. The IL-2 gene from pshuttle/IL-2 was added into adenovirus DNA digested by Pl-scel and I-cel enzyme digestion and incubated for one night. It could integrated into adenovirus DNA and recombinant Ad-IL-2 vector was formed. The positive clone was determined by enzyme digestion and the expression of IL-2 was tested by western blot. 3 Variation of surface phenotype of IL-2 gene-modified dendritic cells.On 6th day of culture, the dendritic cells of healthy and HCC group weretransduced with recombinant adenovirus vector for six hours (MOI=100). TNF-a (200 u/ml) and tumor antigen were supplemented into culture medium in order to promote the immature DCs turn into mature stage. The mature DCs were harvested on day 9 and its membrane molecules were analyzed using flow cytometry. Studied the differences of these active molecules between gene transduced and non-transduced. IL-2 activity of transduced DCs was determined by means of MTT. The changing of immunoactivity of DCs produced by gene transfection was surveyed. 4 To assay the specific cytotoxicity of CTLs activated by transduced DCs.(1) The IL-2 gene modified DCs were harvested on day 9 and coculture with T lymphocytes (DC : T 1 : 4 1 : 20 1 : 100) for four days. T cells proliferation were tested by means of 3H-TdR incorporation. According to that we could calculate the stimulatory index. The supernatant of activated T cells (CTLs) was collected to analyze the IFN- r content with ELISA.(2) HepG2 cells as target cells were harvested and labeled with 200uci Na251CrO4, and the CTLs as effector cell were cocultured with target cells in different ratio (E : T 10 : 1, 30 : 1, 100 : 1). The 51Cr released from the target cells was measured and the percent specific lysis was calculated as follows: 100% x (experimental release-spontaneous release/maximal release-spontaneous release). The difference of lysis capacity caused by gene transfection was investigated.(3) We established the tumor-loaded nude mice mould, and vaccinated CTLs nearby the loaded tumor for immu...