Antitumor Immunotherapy Induced By Tca8113-4-1bbl Cell Cooperatived With Cd28 Mab: An In Vitro Study

Objective To investigate the antitumor response of T cells cooperatively induced by oral cancer vaccine TCV-4-1BBL and CD28 mAb, an in vitro study at the cellular level.Methods 1.Establish clone cell line stablely expressing 4-1BBL:The eukaryotic expression vector pEGFP-4-1BBL was transfected into human oral squamous cell carcinoma cells line Tca8113 by lipofectamine 2000, the transfected cells which stablely expressing 4-1BBL in high level were named as Tca8113-4-1BBL and were selected in medium containing G418400μg/ml, cloned by limited dilution. The expression of 4-1BBL mRNA and protein were tested by RT-PCR and Western blot.2. The tumor cell vaccines (TCV)were obtained by treatment with mitomycin (MMC). the one transfected with 4-1BBL gene is called TCV-4-1BBL, and the untransfected one is called TCV-Tca8113.3.The antitumor ability induced by TCV-4-1BBL cooperated with CD28 mAb:the whole experiment was divided into four groups:①Tca8113 group:T cell+TCV-Tca8113;②Tca8113+CD3 mAb group:T cell+CD3 mAb+ TCV-Tca8113;③Tca8113-4-1BBL+CD3 mAb group:T cell+CD3 mAb+TCV-4-1BBL;④Tca8113-4-1BBL+CD3 mAb+CD28 mAb group:T cell+CD3 mAb+TCV-4-1BBL+CD28 mAb。Each group was cocultivated. After 72 hours, get T cell count by Trypan blue straining, the activity of cytotoxic T lymphocyte (CTL) by Cell Counting Kit-8 and the secretion of cytokines IL-2 and IFN-y.Results 1. The cells line Tca8113 transfected by eukaryotic expression vector pEGFP-4-lBBL showed green fluorescent protein when observed under fluorescence microscope.4-1BBL mRNA and protein was tested by RT-PCR and Western blot analysis.2.The T cell count by Typan blue staining shows group④1.78±0.16, is higher than the other three group:①0.917±0.12,②1.01±0.14,③1.38±0.15, P<0.05, promoted T cell proliferation.3.The cytolytic response of CTL was tested by CCK-8 at 72h after cocultured. Group④59.2±5.1, higher than group②25.7±3.5, group③38.9±3.4 respectively, P<0.05, which promote the activation of T cell. 4. IL-2 were measured by ELISA at 72h in each group. Group④881.2131±78, which much higher than group①56.1619±13, group②176.4235±33, and group③526.1235±67, P<0.05, have promotion in the secreting process of IL-2.5. IFN-y were measured by ELISA at 72h in each group:group④402.81±34, which higher than other groups (②69.83±13,③246.87±24,①37.85±11), have promotion in the secreting process of IFN-y.Conclusions:Oral cancer vaccine TCV-4-1BBL was successfully established. Tca8113-4-1BBL cooperated with CD28 mAb can enhance the immuno-genicity of oral cancer cells, activate the proliferation of T cells, maybe the proliferation in IL-2 and IFN-y is one of its mechanisms.