Study Of Anti-Hepatocellular Carcinoma With Tils Stimulated By S-DCS And Superantigen SEA

Objective:To investigate the killing activities of tumor infiltrating lymphocytes(TILs) that were stimulated by S-DCs (dendritic cells had been sensitized by H22 cells antigen) and superantigen (SAg) staphylococcal enterotoxin A (SEA) on mouse H22 hepatocellular carcinoma cells (H22 cells) in vitro and to observe the antitumor immune capabilities of TILs stimulated by S-DCs and SEA in vivo .To analyze the mechanism of antitumor immune responses and search for a new efficient approach of adoptive transfer immunotherapy on hepatocellular carcinoma.Methods:⑴Bone marrow cell suspensions were isolated from normal mouse,then the granulocytes,T lymphocytes,B lymphocytes,NK cells, mono -cytes and macrophages were removed by treating successively with specific rat anti-mouse CD4,anti-mouse CD8a, anti-mouse CD45R monoclonal antibodies ,rabbit complement buffer and by semi-adhesion of DCs. DCs were cultured with mouse granulocyte macrophage-colony stimulating factor (GM-CSF) and mouse interleukin-4 (IL-4), then a large number of DCs were obtained and DCs were identified.⑵TILs were isolated from the H22 cells tumor by Lymphocyte Separation Medium and then were cultured to amplification in RPMI1640 total culture medium with 10% calf Serum and recombined murine interleukin-2 in vitro.⑶DCs were cultured and then sensitized by H22 cells antigen in vitro.⑷TILs were stimulated by S-DCs and SEA in vitro.⑸Cytoxicity assays experiment in vitro:we divided the effector cells into eight groups,①TILs were stimulated by S-DCs and SEA,②TILs were stimulated by S-DCs,③TILs were stimulated by SEA,④TILs,⑤spleen lymphocytes were stimulated by S-DCs and SEA,⑥spleen lymphocytes were stimulated by S-DCs,⑦spleen lymphocytes were stimulated by SEA,⑧spleen lymphocytes, respectively. Target cells were H22 cells and mouse S180 intestinal cancer cells.The ratio of the effector cells to the target cells was 10:1. The killing activities of the effector cells to target cells were detected by MTT experiment, then compared with them and statistics analyse.⑹Adoptive treatment experiment in vivo: it included ten groups,twenty mouse bearing tumor in each group of nine,treated by TILs stimulated by S-DCs and SEA,TILs stimulated by S-DCs, TILs stimulated by SEA,TILs, spleen lymphocytes stimulated by S-DCs and SEA, spleen lymphocytes stimulated by S-DCs, spleen lymphocytes stimulated SEA, spleen lymphocytes, and normal saline through trail vein respectively. Twenty normal mouse in the last group treated by normal saline through trail vein.The activities of splenocytes NK, LAK, CTL activity and the serum TNF of mice after treatment were assayed respectively and inhibition of the progerssion of tumor and pathologic changes of tumor of mice bearing tumor were assayed as well.Results:(1)The result of cytoxicity assays experiment:①The killing rate in each effector cells groups on H22 cells was obviously higher than the corresponding control groups on S180 cells respectively. The distinction of killing rate showed statistical significances (p<0.01).②The killing activity of TILs on H22 cells [killing rate: (51.21+3.00)%] was much higher than spleen lymphocytes on H22 cells [killing rate: (19.60+2.17)%],the distinction of killing rate showed statistical significances (p<0.01).The killing rate in each effector cells groups that difference manner stimulated TILs on H22 cells were obviously higher than the corresponding each effector cells that difference manner stimulated spleen lymphocytes on H22 cells respectively. The distinction of killing rates showed statistical significances (p<0.01).③TILs stimulated by SEA had higher killing activity on H22 cells [killing rate: (62.70+2.42)%] than the killing activity of TILs on H22 cells[killing rate: (51.21+3.00)%],TILs stimulated by S-DCs had much higher killing activity on H22 cells [killing rate: (75.23+2.71)%],TILs stimulated by S-DCs and SEA had the highest killing activity [killing rate: (85.66+2.62)%]in all groups,the distinction of their killing rate showed statistical significances (P<0.01).(2)The result of adoptive treatment experiment in vivo :①the activities of splenocyte NK,LAK,CTLand serum TNF of mice bearing tumor can be induced to enhance obviously by TILs stimulated by S-DCs and SEA ,the killing rates were (34.23+1.52)% ,(35.50+1.89)%,(39.83+1.55)% respectively and serum TNF wa(s46.24+1.26)U/ml.They were little higher than normal groups and had significant difference comparing with TILs stimulated by S-DC group, TILs stimulated by SEA group,TILs group, spleen lymphocytes stimulated by S-DCs group and SEA group, spleen lymphocytes stimulated by S-DCs group, spleen lymphocytes stimulated SEA group, spleen lymphocytes group, and normal saline group.②Tumor progression of mouse bearing tumor: The proliferation of tumor were inhibited significantly in mouse bearing tumor after treatment by TILs stimulated by S-DCs and SEA , tumor size,tumor weight and inhibiting tumor rate were(0.402±0.10)cm,(0.394±0.11)g and 79.39% respectively.The distinction was significant compared with other treated groups(P<0.01).③Pathological inspection observed a large amount of lymphocytec infiltrated and extensive necrosis in tumor tissues in the group treated by TILs stimulated by S-DCs and SEA , and that showed statistical significances compared with other treated groups(P<0.01).Conclusion: TILs stimulated by S-DCs and SEA has the most efficient and specific killing activity on H22 cells in vitro,and can improve antitumor immune responses specificly and nonspecificly in mouse bearing tumor,and obviously inhibite tumor progression as well.These results are to expect strongly to provide a new and effective way of clinical treatment on hepatocellular carcinoma.