The Acquisition And Expansion Of Vγ9Vδ2-T Cells And The Preliminary Exploration Of Their Antitumor Effects

Objective:Vγ9Vδ2-T cells comprise a large proportion of the peripheral blood γδ-T cells of humans and possess a potent anti-tumor activity to a variety of cancers.It is a promising method to adapt the Vγ9Vδ2-T cells derived from healthy individuals in cancer patients and this cellular immunotherapy may be translated into clinical accompanied with extensive benefits.However,although Vγ9Vδ2-T cells have been demonstrated to be expanded in vitro,obstacles remain in acquiring sufficient Vγ9Vδ2-T cells with demanded purity as well as effectively anti-tumor activity in vivo.This study intends to probe the effects of diverse concentrations of Zoledronic acid(Zol)and interleukin-2 on the expansion of Vγ9Vδ2-T cells in vitro,initially to establish a complete basic culture system of Vγ9Vδ2-T cells,and to explore the role of CD137 in the stimulation of the anti-tumor activity of Vγ9Vδ2-T cells in vitro and in vivo.Methods:1.Zol and IL-2 were applied to amplify Vγ9Vδ2-T cells from peripheral blood mononuclear cells,interleukin 15 was added in the protocol to enhance the cytotoxicity of Vγ9Vδ2-T cells.Evaluate the effects of different concentrations of Zol and IL-2 on the expansion of Vγ9Vδ2-T cells,and preliminarily establish a comprehensive basic Vγ9Vδ2-T cell culture plan.2.Vγ9Vδ2-T cells were co-cultured with tumor cells(Raji,Farage,Hep G2,Kyse-150,NCI-H69,A549)and evaluated the killing effect of Vγ9Vδ2-T cells on tumor cells.3.CD137 was applied at different times and diverse concentrations in the culture of Vγ9Vδ2-T cells,and the effects in the stimulation of Vγ9Vδ2-T cells were evaluated.4.The culture medium of Vγ9Vδ2-T cells was collected and centrifuged,and obtained the supernatant.IFN-α ELISA kit and IFN-γ ELISA kit were used to quantitatively the expression levels of IFN-α and IFN-γ in the culture medium of the groups of control and CD137 stimulation.5.Soft agar clone was used to clone Vγ9Vδ2-T cells in vitro and observe the formation of Vγ9Vδ2-T cells.6.C57BL/6 mice were injected intraperitoneally with Vγ9Vδ2-T cells,observe whether there is a local stimulation reaction,toxic reaction,allergic reaction,and weight change after the mice are injected with T cells,and evaluate the safety of Vγ9Vδ2-T cells.7.Construct a tumor transplantation model,inoculate tumor cells,and treat with expanded Vγ9Vδ2-T cells in vitro.Compared the tumor growth rate,body weight change,etc.between the control and experimental mice groups,and evaluated the antitumor activity of Vγ9Vδ2-T cells.Results:1.During the culture of Vγ9Vδ2-T cells,the Vγ9Vδ2-T cells obtained by stimulation with high concentration of Zol(50μM)at Day0 were three times as high as those in the low concentration(10μM)stimulation group at Day14.However,the cells gradually decreased and died when the cell culture medium was changed at Day0,Day3,and Day6 and stimulated with high concentration of Zol.2.The cell colonies of Vγ9Vδ2-T cells are more larger in the group of higher concentration IL-2(1600IU/ml for the first stimulation and 400IU/ml for the further stimulation)than the group of lower concentration IL-2(400IU/ml for the first stimulation and 100IU/ml for the further stimulation).3.The Vγ9Vδ2-T cells,on the 14 th day of expansion,were selected to co-incubate with different tumor cells(Raji cells,Farage cells,NCI-H69 cells,A549 cells,Kyse-150 cells,Hep G2 cells),and the effect-target ratio is 10:1.The results showed that Vγ9Vδ2-T cells could inhibited the proliferation of these tumor cells.4.The Vγ9Vδ2-T cells expanded in vitro were counted at different times,there was no significant difference in the number of Vγ9Vδ2-T cells in the 5 ng/ml CD137 stimulated group compared with the control group,But under the 5 ng/ml CD137 stimulation condition,the expression level of cell active factors in the culture medium and the efficiency of killing of the tumor cells were higher than the other experimental groups,killing efficiency of Raji cells: 22.70 ± 4.18% in control,14.00 ± 0.69% at 2 ng/ml stimulation(P <0.001),27.10± 2.67% at 5 ng/ml stimulation(P=0.044),18.10 ± 0.63% at 10 ng/ml stimulation(P=0.020),11.50 ± 0.58% at 20 ng/ml stimulation;Killing efficiency of NCI-H69 cells:17.60 ± 1.27% in the control group,11.50 ± 0.86% at 2 ng/ml stimulation(P <0.001),19.70 ± 2.04% at 5 ng/ml stimulation(P=0.037),17.60 ± 0.72% at 10 ng/ml stimulation(P=0.982),10.70 ± 0.64% at 20 ng/ml stimulation(P <0.001).The results showed that the anti-tumor activity of Vγ9Vδ2-T cells was enhanced in vitro when stimulation with 5 ng/ml concentration of CD137.5.CD137(5ng/ml)was intervened in vitro from the 0th/6th day of Vγ9Vδ2-T cell expansion,and co-cultured with tumor cells at 10:1 on the 14 th day of Vγ9Vδ2-T cell expansion,the killing efficiency of the control group on tumor cells was Raji 22.70±4.18%,NCI-H69 17.60±1.27%,and the killing efficiency of Day0 stimulation group on tumor cells was Raji 18.60 ± 1.44%(P=0.021),NCI-H69 17.18 ± 1.19%(P=0.530),the killing efficiency of T cells in Day6 stimulation group on tumor cells was: Raji 27.10 ± 2.67%(P=0.044),NCI-H69 19.70 ± 2.04%(P=0.037).The results showed that T cells obtained from stimulation on the 6th day had a higher killing effect on tumor cells.6.The clone formation was 0 both in the group of 0 ng/ml or 5ng/ml CD137 stimulation of Vγ9Vδ2-T cells.7.After intraperitoneal injection of Vγ9Vδ2-T cells(0ng/ml CD137 stimulation group,5ng/ml CD137 stimulation group)into mice,there was no significant difference in the weight of mice in the experimental group and control group,and no local irritation or allergic reaction occurred.8.The tumor growth rate of mice in the 0ng/ml CD137 stimulated Vγ9Vδ2-T cell treatment group was slower than that in the PBS treatment group,and the tumor growth rate of mice in the 5ng/ml CD137 stimulated Vγ9Vδ2-T cell treatment group was significantly slower than that in the 0ng/ml CD137 stimulated Vγ9Vδ2-T cell treatment group and PBS treatment group.The results proved that Vγ9Vδ2-T cells had an anti-tumor effect in vivo,and CD137 stimulation could enhance the anti-tumor of Vγ9Vδ2-T cells.Conclusions:Zol combined with IL-2 and IL-15 can cultivate and obtain a large number of Vγ9Vδ2-T cells with strong activity.CD137 stimulation can enhance the activity of Vγ9Vδ2-T cells after intervening in culture,and the anti-tumor activity of tumor cells in vitro is also improved.Since Vγ9Vδ2-T cells can be massively expanded in vitro,they may become a new method of cancer immunotherapy.