| Ativated T lymphocytes are a critical component of the immune responseto tumors.They are sufficient to protect against tumors and can eliminate evenestablished cancers in murine tumor models and in humans. At least two sign-als are required to activate naive T lymphocytes. The first signal involves theinteraction of the MHC-peptide with the TCR and CD4 or CD8 receptors onT-lymphocytes. The second signal involves the interaction between costimulatoryfactors such as members of B7 family on the antigen presenting cells (APCs)and CD28 or CTLA-4 on T lymphocytes. Once this second signal is accompli-shed,the first signal will suffice to maintain T-lymphocyte activity. In responseto certain inflammatory cytokines,APCs express costimulatory molecules suchas B7,which is normally expressed on macrophages cells and B lymphocytesand reacts with CD28 to activate T cell. In the absence of the appropriate co-stimulatory signals,engagement of the T cell receptor itself typically leads tounresponsiveness,anergy or apoptosis of the antigen specific T lymphocytes.The human breast carcinoma cells often fail to express B7,thus, the antigenpresented in its absence will be ignored by the immune system. Another impo-rtant aspect of immunity is the recruitment and accumulation of lymphocytesinto taget tissues. Some chemokine family members are chemotactic for selectedpopulations of immunocytes,and may exert indirect effects on antitumor byinhibit angiogenesis. Genetic modification of many types of tumor cells,whichexpress either costimulatory molecules,such as B7-1,or chemokines,such asMIP-lα,can increase the immunogenicity. To augment antitumor effect in vivo, the combination treatment of breast carcinoma cells with costimulatory moleculegenes and chemokine genes mediated by retrovirus was investigated.The use of retroviral vectors for gene therapy has drawn much attentionand currently is the choice for the transferral of therapeutic genes in a varietyof approved protocols both in the U.S.A and in Europe. We have generatedtwo retroviral constructs that contained one destination gene and one resistancemarker respectively: B7-1 gene or MIP-1αgene,G418 resistance or puromycinresistance. We transfected SHZ-88 cell line with MIP-lαor B7-1 gene respecti-vely. Moreover,we also transfected the tumor cells with both MIP-lαand B7-1genes. By one or two drug screening test,some positive clones,which expressMIP-1αand (or) B7-1,has been obtained in 14 days. None of the transfectants,including the multi-gene tranfectant,was demonstrated that altered grow rateand cell appearance in vitro. The expression of MIP-1αand B7-1 mRNA ingene-transfected SHZ-88 cell was showed by RT-PCR. The expression of B7-1on gene-transfected SHZ-88 cell was showed by flow cytometry; While the ex-pression of MIP-lαon gene-transfected SHZ-88 cell was confirmed by immu-nohistochemistry. When mB7-1 transfected cells were used as stimulators ofallogeneic spleen lymphocytes in mixed lymphocyte-tumor culture(MLTC),MTTassay indicated that T-lymphocyte proliferations were significantly increased.Chemotaxis assay demonstated that mMIP-lαmodified cells can secreted subst-antial levels of chemokine activity for activated rat spleen lymphocytes. Thetumor volumes indicated the injection of MIP-lα-transfected or B7-1-transfectedSHZ-88 cells subcutaneously(s.c.) could inhibit the growth of SHZ-88 cellsand prolonged the animal tumor-bearing life span in vivo compared with thecontrol group (P<0.05). The inhibitory effect was enhanced when the MIP-lα+B7-1 co-transfected SHZ-88 cells were used (P<0.05). Flow cytometry showedthat the level of CD4+,CD8+ and CD4+ / CD8+ ratio in the MIP-lα+ B7-1 gr-oup was significantly higher than that of the single gene-transfected or control group(P<0.05);EILSA showed that the content of serum IL-2 and IFN-γinMIP-lα+B7-1 group was significantly higher than that in the single gene-trans- fected or control group (P<0.05). HE stain showed more lymphocyte infiltrationwere found in the inoculationsites of SHZ-88 / MIP-1α+B7-1 cells than in that ofMIP-lαor B7-1-transfected SHZ-88 cells. These results suggest that the comb-ination of B7-1 gene and MIP-1αgene can augment antitumor effect in vivo.The antitumor effects are not significant difference after gene-transfected cells wereinjected intumor center or opposite side axillary fossa.(P>0.05).The main conclusions of the in vitro and in vivo experimental studies areas follows:1. A new rat breast cancer cel1 strain,SHZ-88 / mMIP-1α+mB7-1, whichcould co-express MIP-1α+B7-1 gene stably,was established successfully,pos-sessing biologic activity in vitro;2. The injection of MIP-lα+B7-1 gene transfe-cted SHZ-88 cells could induce stronger antitumor effect than that of B7-1 or MIP-1αgene transfected SHZ-88 cells in vivo.
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