Killing Activity Of γδT Cells Against Human Hematologic Neoplasms Cells In Vitro

Hematologic neoplasms is a general term for a series of malignant malignant of hematology system,including leukemia,multiple myeloma,myelodysplastic syndromes,lymphoma and malignant histiocytic disease.The incidence of hematologic neoplasms is high in China.As combination chemotherapy regimen and supportive therapy continu to improve,the initial clinical remission rate and survival rate of hematologic neoplasm are increasing significantly.Despite achieving Initial clinical remission,hematologic neoplasm is still associated with recurrance.Especially mortality of acute leukemia is high and long-term survival rate is low.While some cases fail to achieve clinical remission,the long-term survival rate is lower.The treatment effect of relapsed and refractory cases is still limited,and there is no ideal treatment.Traditional therapies such as surgery,radiotherapy,chemotherapy and targeted therapy are aimed at tumor cells and tissues.Traditional therapies can eliminate tumor cells partially,but can not eliminate all tumor cells.So those cases are inevitable recurrence.Tumor immunotherapy is aimed at the immune system,by improving eliminate tumor capacity of the body’s immune system to achieve the purpose of treatment of tumor.In 2013,Science magazine ranked cancer immunotherapy as the top ten breakthroughs in the year.Tumor immunotherapy offers hope for cancer treatment.Adoptive immune cell therapy is expansion and activation autologous or allogeneic immune cells in vitro and reinfusion to the patients,utilizing activated immune cell antitumor activity to eliminate the tumor cells in patients.At present,T cells,NK and DC cells are commonly used in adoptive immunotherapy.T cells are deemed to be the only immune cells that can specifically recognize and kill tumor cells.According to the different expression of TCR,T lymphocytes can be divided into αβT cells and γδT cells.γδT cell is a T cell subset which express TCR γδ.γδT cells account for only 1-5% of peripheral T lymphocytes,but in the organization can be as high as 20-50%.γδT cell is the body’s natural immune cell.γδT cell plays anti-tumor,anti-viral infection,anti-bacterial infection and immune surveillance,promoting the secretion of Ig A,Ig G and Ig M of immature B cells,regulation of humoral immunity and activating immature DC cells in vitro.γδT cells recognize a variety of tumor associated antigens in non-MHC-restricted manner,and kill tumor cells in the following ways: inducing apoptosis of tumor cells via protein ligand pathway induced Fas-Fas L and TRAILR;secreting a large number of cytokines which act on tumor cells and their microenvironment and directly or in cooperation with other immune cells to kill tumor cells;killing tumor cells by means of ADCC and perforin granzyme B;through some membrane surface receptors such as Fcγ R,playing the role of cytotoxicity by means of ADCC;playing a role of antigen presentation as APC.A number of clinical trials have been performed using γδT cells to treat tumors,and progress has been made in the treatment of colorectal cancer,renal cell carcinoma,non-small cell lung cancer,breast cancer and pancreatic cancer.Those studies have found that γδT cells have cytotoxic effect against a variety of tumor cells and have achieved good efficacy and safety in clinical trials.There are three advantages of applying γδT cells in the treatment of hematological malignancies.1.Activated γδT cells have high cytotoxicity against hematologic malignancies cells.2.γδT cells can secret a large number of cytokines and present antigens to cooperate with other immune cells to kill hematologic malignancies cells.3.Patients with hematologic malignancies are often associated with infection due to poor immunity or immunity defects,but γδT cells are innate immune cells which can eliminate tumor cells and resist virus and bacterial infection.AIM: The aim of this study is to establish culture system of γδT cell,to explore cytotoxicity of γδT cells against different human hematologic neoplasms cells and to explore some mechanisms that affect the cytotoxicity of γδT cell,which could provide experimental evidences for cellular immunotherapy for hematologic malignancies.CONTENTS: The research consists of two parts.1.The γδT cell culture system was established by optimizing the culture scheme of zoledronic acid(Zol)combined with IL-2.The effect of culturing γδT cells was compared with two kinds of serum-free T cell culture medium.2.Those are detected: the cytotoxicity of γδT cells against a variety of hematologic malignancies cells,which include Jurkat,THP-1,HL-60,K562,Raji,U-937 and RPMI-8226 cells;the level of IFN-γ and TNF-α secreted by γδT cells;the effect of prolonged incubation time with target cells on the killing activity of T cells against target cells;the effect of prolonged incubation time with target cells on the killing activity of γδT cells against target cells;the effects of zoledronic acid and mevastatin on γδT cells killing tumor cells;the cytotoxicity of γδT,NK and CIK Cells against K562 cells.METHODS: 1.To optimize the culture medium of zoledronic acid combined with IL-2,and to establish the cell culture system of γδT cells by using RPMI-1640 medium.On this basis,the effect of two kinds of serum-free T cell culture medium Op Tmizer and X-VIVO 15 culture medium on the culture of γδT cells in vitro was compared,and the effects of serum addition on the proliferation of γδT cells in serum-free medium were compared.2.The Jurkat,THP-1,HL-60,K562,Raji,U-937 and RPMI-8226 cells were cultured in vitro and observed under inverted microscope.To compare of the killing activity of T cells against Jurkat,THP-1,HL-60,K562,Raji,U-937 and RPMI-8226 cells and effects of different effect target ratio on the killing of γδT cells,Those cells were incubated with γδT cells for 4 h,the effective target ratio being 10:1 and 20:1,and then detected by flow cytometry.γδT cells and K562 cells were incubated for 48 h with the effective target ratio 10:1,and the levels of IFN-γ and TNF-α were detected by flow cytometry at 0,4,16,24,36 and 48 h,respectively.The HL-60 and K562 cells were co-incubated γδT cells for 4 h and 20 h by effector target ratio of 10:1 respectively,and the co-incubation time on cytotoxicity of γδT cells against HL-60 and K562 cells was detected by flow cytometry.After different concentrations of zoledronic acid pretreated K562 cells respectively,γδT cells were co-incubated with K562 cells for 4 h by effector target ratio of 10:1,and the effects of different concentrations of zoledronic acid on cytotoxicity ofγδT cells against K562 cells was detected by flow cytometry.K562 cells were pretreated with atorvastatin(5 mol/L)and then co-incubated with γδT cells for 4 h by effector target ratio of 10:1,and the effect of fluvastatin on the Killing activity of γδT cells against K562 cells was evaluated by flow cytometry.After signing the informed consent,PBMC were generated from peripheral blood of 4 randomly selected patients with lymphoma.γδT and NK and CIK cells were amplificated in vitro by different culture system.Those cells were incubated with K562 cells for 4 h at effect target ratio 5:1,10:1 and 20:1 respectively.The killing activity of γδT,NK and CIK cells against K562 cells was compared by flow cytometry.RESULTS: 1.The γδT cells were successfully amplified from peripheral blood in vitro by optimizing the culture scheme of zoledronic acid combined with IL-2.The effect of Op Tmizer medium on culture of γδT cells in vitro was better than that of X-VIVO 15 medium(P<0.05).Appropriate addition of serum can improve the efficiency of serum-free medium to amplify γδT cells(P<0.05).2.The γδT cells showed obvious cytotoxicity activity against all of Jurkat,THP-1,HL-60,K562,U-937 and RPMI-8226 cells(P<0.05)but showed dull cytotoxicity activity against.With extending the co-incubation time with K562 cells,the level of IFN-γ secreted by the γδT cells increased as a time-dependent manner,in addition,the level of TNF-α secreted by the γδT cells gradually increased after 8 h of the co-incubation.After co-incubation of the γδT cells with K562 and HL-60 cells,the expression level of CD107 a molecule on the γδT cells was significantly up-regulated(P<0.01).Prolonging the time of co-incubation of γδT cells with K562 and HL-60 cells,the killing activity of γδT cells against K562 and HL-60 cells was significantly enhanced(P<0.01).Zol increases the cytotoxicity of γδT cells against K562 cells,while mevastatin attenuates that of γδT cells.from γδT,NK and CIK cells are successfully cultured in vitro,which came from peripheral blood of 4 Patients with B cell lymphoma.When the effect target ratio was 5:1,there was not a statistical difference between cytotoxicity activity of γδT,NK and CIK cells against K562 cells(P>0.05).When the effect target ratio was 10:1 or 20:1,there was not a statistical difference between cytotoxicity activity of γδT cells and NK cells against K562 cells(P>0.05),but cytotoxicity activity of γδT cells against K562 cells was higher than that of CIK cells(P<0.01).CONCLUSION: The γδT cells were successfully amplified from peripheral blood from healthy volunteers in vitro by optimizing the culture scheme of zoledronic acid combined with IL-2.The effect of Op Tmizer medium on culture of γδT cells in vitro was better than that of X-VIVO 15 medium.Appropriate addition of serum can improve the efficiency of serum-free medium to amplify γδT cells.γδT cells showed obvious cytotoxicity activity against several hematologic neoplasms cells.With extending the co-incubation time with K562 cells,the level of IFN-γ secreted by the γδT cells increased as a time-dependent manner,in addition,the level of TNF-α secreted by the γδT cells gradually increased after 8 h of the co-incubation.After co-incubation of the γδT cells with K562 and HL-60 cells,the expression level of CD107 a molecule on the γδT cells was significantly up-regulated.Prolonging the time of co-incubation of γδT cells with K562 and HL-60 cells,the killing activity of γδT cells against K562 and HL-60 cells was significantly enhanced.Zol increases the cytotoxicity of γδT cells against K562 cells,while mevastatin attenuates that of γδT cells.There was not a statistical difference between cytotoxicity activity of γδT cells and NK cells against K562 cells,but cytotoxicity activity of γδT cells against K562 cells was higher than that of CIK cells.In summary,γδT cells,which cultured in vitro by zoledronic acid combined with IL-2,showed higher cytotoxicity activity against hematologic neoplasms cells in vitro.After the incubation with target cells,the killing activity of γδT cells was increased.Improving the effect target ratio and prolonging the co-incubation time with target cells,can improve the killing activity of γδT cells against target cells.Zoledronic acid enhanced the killing activity of γδT cells against K562 cells,while atorvastatin decreased.Those could provide experimental evidence for cellular immunotherapy of hematologic neoplasms.