The Study On The Basic Characteristics And The Anti-tumor Effects Of γδ T Cells Cultured In Vitro

Objectives γδ T cells play a killing activity does not require specific antigen stimulation,no major histocompatibility complex(MHC)restrictions,has good anti-tumor activity and a variety of biological functions,is a powerful tool for tumor Cellular immunotherapy.Studies have shown that using in vitro amplification of γδ T cells to treat a variety of solid tumors have achieved good therapeutic effect,but using different methods of amplification γδ T cells have a quite different antitumor activity,directly affects the clinical therapeutic effect.Therefore,it is significant to establish and optimize the large-scale culture techniques of γδ T cells,to obtain the γδ T cells which has high anti-tumor activity and can be used in the clinical treatment.In this study,we will establish and optimize the in vitro amplification method of γδ T cells from peripheral blood,and evaluate the anti-tumor activity and safety of the expanded γδ T cells in vitro and in vivo.Methods1)Peripheral blood mononuclear cells were isolated from 10 healthy volunteers.2)GT-T551H3 medium supplemented with stimulating factors,such as phytohaemagglutinin,zoledronic acid,interleukin-2 and interleukin-7.According to the multiplication and purity of γδ T cells in vitro,the optimal combination,concentration and adding time of stimulating factors were explored,and the in vitro amplification process of γδ T cells was established.3)Using the optimized culture system respectively to culture the γδ T cells isolated by immunomagnetic beads and peripheral blood mononuclear cells(PBMC)in vitro.The growth state and speed of the cells were observed,and the purity of γδ T cells was detected.The total number of cells was calculated by cell count.4)The target cells,such as SK-MES-1,K562,A549 and Ho8910 cells,were labeled with red fluorescent dye.The proliferation of γδ T cells in vitro was used to carry out the killing experiment for 4 hours at different effect-target ratios.The cytotoxic activity of γδ T cells was detected by flow cytometry.5)The in vivo antitumor activity of expanded γδ T cells was evaluated in nude mice model of lung cancer transplantation.Mice in experimental group were treated with expanded γδ T cells in vitro,and mice in control group were treated with aseptic PBS solution.The tumor growth rate was compared between the two groups.6)The acute toxicity of γδ T cells was detected by C57 BL / 6 mice.The mice in the experimental group were injected with expanded γδ T cells in vitro,and the control mice were injected with aseptic PBS solution intraperitoneally.The survival and acute toxicity of the mice were observed.7)The contamination of exogenous factors in γδ T cells was detected by PCR method and culture in vitro,and the clone forming ability of γδ T cells was detected by soft Agar cloning kit.Results The optimized γδ T cell culture medium was serum-free medium GT-T551 H3 mediumsupplemented with the following concentration of stimulating factor: 200 IU/ml IL-2,10 μ g /L,IL-7,1.0 μ g/ml phytohaemagglutinin,1.0 μg/ml Zoledronate,0.4%autologous plasma.After cultured at 37 ℃ for 14 to 16 days,γδ T cells expanded more than 1000 times and the total number of cells was more than 1.0 × 10 10.The proportion of CD3+ γδ TCR+ cells was more than 90.There were no contamination of bacteria,fungi,mycoplasma and viral factors.γδ T cells cultured in vitro were highly cytotoxic to lung squamous cell carcinoma cell line SK-MES-1,ovarian cancer cell line Ho8910,lung adenocarcinoma cell line A549 and chronic myeloid leukemia cell line K562.The killing efficiency of γδ T cells was more than 65%(the effective target ratio was 50:1).The tumor volumes of the experimental group and the control group were(828.99±61.05)mm3 and(1723.51 ±84.30)mm3,respectively.The difference was statistically significant(P < 0.05).There was no acute adverse reaction in the mice infused with γδ T cells.Conclusion The γδ T cells cultured in this study have sufficient quantity,high purity,strong killing activity,good anti-tumor activity and safety in vivo and in vitro,and have killing activity to lung cancer,ovarian cancer and leukemia tumor cells,which meet the requirements of clinical application.Because of its simple and feasible operation and low cost,it can be expanded in vitro and has a good prospect of clinical application.