Experimental Study On Tumor Infiltrating Lymphocytes Adoptive Immunotherapy Of Hepatocellular Carcinoma

Purpose: The present study probes to access the effect on the proliferation and cytotoxicity of the tumor infiltrating lymphocytes induced by interleukin-12 and activated by dendritic cells loaded with tumor antigen and to evaluate the possibility that IL-12 and DC enhance the specific cytotoxic activity of TILs to tumor cells. Based on these to find a way to increase the effect of adoptive immunotherapy by TILs on patients with PHC.Methods: We derived TILs from tumors resected from 5 cases of primary hepatic cancer. They were cultured in complete culture medium in which IL-12 (5pg/ml) was added in with or without IL-2(100U/ml).The culture medium added with IL-2 in was as contrast group. The cytotoxic activity , augmentation ability and cytokine production were tested separately 10 to 14 days after the culture. CD3 , CD4, CD8 and CD16+56 were analysed by Flow cytometry (FCM). In the course of the culture, the mononeuclecytes divided from the peripheral blood cells of patients with PHC induced by GM-CSF(800U/ml) andIL-4(500U/ml) in vitro. Then autologous tumor cells were given to stimulate them to get DC loaded with tumor antigens and one week later, cultured with TIL for 7 days the amount of DCs were accounted.In addition, the expression on surfaces of TILs tested by FCM before and after mixed culture and the cytotoxic activity and cytokine production were tested. The results expressed with (X?SD) and the statistical analyses were performed by using Student's t test in two groups and One-Way ANOVA in more groups. The used software was SPSS 8.0.Results: 1.The cytotoxic activities in IL-12+IL-2 group and IL-12 group to autologous hepatic cancer cells were higher significantly than that in IL-2 group (P<0.001; P<0.01) as to SK-hep-1 and K562 tumor cells, the results were the same (P<0.05). 2. In IL-12+IL-2 group the CD3+ , CD4+ and CD8+ rates were higher significantly than that of IL-2 group (P<0.01), but differences of CD16+56+ rate had no siginificance by using Student's t test. 3. In IL-12+IL-2 group the augmentation activities of TILs were higher than that in IL-2 group (P<0.05). 4. Comparing with TILs cultured in complete culture medium contained IL-2, the augmentation activities of TILs mixed with DC was enhanced (P<0.01). 5. The secretions of IFN- ? and TNF- ? from TILs mixed with DC also were added (P<0.01). 6. The cytotoxic activities of the TILs stimulated with DC to autologous hepatic cancer cells were higher than to SK-hep-1 and K562 tumor cells (P<0.01 ;P<0.05).Conclusion: 1. As an inducer IL-12 induced TILs of hepaticcancer and DC loaded with tumor antigen activated TILs, resulted in enhanced obviously specific cytotoxicity and proliferation of TILs; 2. Small dose of IL-2 added with IL-12 may highly increase the anti-tumor action and there be a cooperation action. 3.TILs induced by IL-12 and DCs have higher cytotoxic activities to autologous hepatic cancer cells than to SK-hep-1 and K562 cells, which suggest higher specific roles. 4.The increased cytokines such as IFN- ? and TNF- ? can enhance the anti-tumor function of TIL.These results provide some new supports for us to raise the treatment effects of adoptive immunotherapy with TILs induced by IL-12 and activated by DC on tumors and they will give us an instruction on clinical applications.