Large Scale Amplication Of The Tumour Infiltrating Lymphocytes From Hepatoma Ex Vivo And Analysis Of Their Biological Characters

Research ContextThe earliest report concerning tumor infiltrating lymphocytes (TIL) immunotherapy was in 1988 when Rosenberg and his collegues first demonstrate that using TIL for the immunotherapy of patients with metastatic melanoma can make tumor regression.More and more researches concerning TIL were emerged afterwards and the most remarkable therapeutic effect appeared in melanoma patients.Rosenberg and his groups had published in Science magzine that lymphodepletion prior to ACT can improve the objective response to 50% in 2002.After that,in 2005,they had reported that the objective response can increased to 70% after they mended their disporal method.The objective response of traditional treatment on metastatic melanoma including IL-2 and dacarbazine was only 15%-25% from which make the superiority of TIL therapy more obvious.Like the metastatic melanoma,hepatic carcinoma is also kinds of high immunogenicity malignant tumor.There is no effective therapy in advanced stage except being operated in earlier period. That is why people endeavor to cure hepatic carcinoma through immunotherapy. However,the using of TILs for therapy developed slowly.It's partially because there is no mature methods for culturing and expanding TILs,also,the instable technique restrict the clinical application.In view of the above reasons,the main purpose of this project is to explore new technique for culturing and expanding TILs,laying the foundation for making the TILs use for clinical therapy. Purpose:1.Explore the new methods for Large scale amplication of the tumour infiltrating lymphocytes from hepatoma ex vivo.2.Study the phenotypes of the TILs,detect their abilities of secreting the cytokines and analyse their fuctions.3.Observe the impact of different irridiation (0,1,2Gry) on TILs phenotypes and fuctions.Methods:The specimen of primary liver cancer come from the excitional tumors.These solid tumor fragments were immersed in a mixture of collagenase,hyaluronidase,and DNAse in serum-free RPMI1640, and incubated overnight with gentle agitation.The single -cell slurry was passed through sterile wire mesh to remove undigested tissue chunks.The TILs were seperated by density gradient centrifugation and planted in different culture essels.The planting TILs of every specimen were irradiated by different doses (0,1,2Gry).After that the TILs were expanded by two-step culture method. Molecular phenotype such as CD3,CD56, CD4,CD8,CD25, Fox-p3, CD27,CD28 of generated TILs were tested by flow cytometry and the quantitation of basic cytokine secretion and following cytokine secretion after stimulated by PHA of (IFN-γ,TNF-α, IL-4,IL-5,IL-10) were detected by Cytometric Bead Array(CBA) essay.In addition to analysing the impact of secretion capability on the TILs'fuction,there were also a comparition between the effection from different irriadiation doses on TILs'cytokine secretion fuction.Result:1. There were eighteen TILs samples could reach the 1010 order of magnitude through two-step culture method.2. The proportion of CD3+TILs was 95.7%±7.4%,as in the CD3+CD4+TILs and CD3+CD8+TILs these were 32.5%±19.6% and 62.5%±29.1% respectively.The proportion of CD3+CD56+ TILs was 28.5%±11.7%.The expression of CD27 couldn't be detected and the proportion of CD28 were 71.1%±28.8%.The expression of CD4+CD25+ TILs and CD4+Fox-p3+ TILs in CD3+ T cells was 2.1%±0.6% and 2.6%±0.9%.3. The everage quantitation of cytokine secretion of IFN-γ, TNF-α,IL-10,IL-5 and IL-4 in expanded TILs were 2147.9±406.6pg/ml,3.6±1.3/ml,13.9±4.6/ml,2265.1±712.4 pg/ml,21.6±5.9/ml respectively.The corresponding quantitation of cytokine secretion after stimulated by PHA were 12019±3150.6pg/ml,6634.0±1763.5pg/ml, 29.2±3.7pg/ml,8624.3±609.1pg/ml and 112.4±34.5pg/ml.4. The different TIL samples had different response to irriadiation.The eight of ten irradiated TIL which is mainly CD8+T cells could appear regular response when detected by FCM.The detail is CD3+CD8+T cells, CD3+CD28+T cells could decrease following increasing dose of irradiation.In the eight TILs, there were six TIL'CD3+CD56+T cells had a regularly decreasing trend and the other two had not.5.As detected by CBA,the secretion of cytokine according to the different dose of irriadiation had regular either.It's probably confested that the quatity of cytokine secretion are related to the number of CD3+CD56+T cells.But it stills need more evidence to confirm this conclusion.