The Study Of γδ T Cells Regulating Proliferation,apoptosis And Autophagy Of Myeloma Cells

Objective:To investigate the effect ofγδT cells on the proliferation,apoptosis and autophagy of multiple myeloma cells.Method:Peripheral blood mononuclear cells were isolated from healthy adults by using lymphocyte Ficoll centrifugation method,and stimulated with 1μM Zoledronate(Zol),in combination with 400 IU/m L Interleukin(IL)-2.After 1,6 and 10days culturing,γδT cells were harvested and collected.Then,γδT cells purity analysis was conducted by flow cytometry.γδT cells were collected at d 10 of culture.Then we used transwell inserts to establish co-culture system,withγδT cells in the upper compartment and RPMI 8226 or U266 cells in the lower compartment at different ratios.With the intervention ofγδT cells,these co-cultured myeloma cells were collected and the changes of cell proliferation was detected by CCK8 assay.The cell cycle distributions and apoptosis rates of co-cultured myeloma cells,which were intervened byγδT cells,were measured by flow cytometry.The expression of cell cycle-related(Cyclin D1,CDK4 and CDK1),apoptosis-related(Bax,cleaved caspase3and Bcl-2)and autophagy-related proteins(LC3B,P62,PI3K,AKT,P-AKT,P-m TOR,Beclin-1 and AMPK)of co-cultured myeloma cells were detected by Western blot.Culture supernatant of the co-cultured myeloma cells were collected after 24 h of co-cultured withγδT cells,and the concentrations of interferon(IFN)-γand tumor necrosis factor(TNF)-αwere detected,using ELISA.Results:γδT cells can be expanded with Zol and IL-2 in vitro,and the percentage of CD3~+gammadelta~+cells was as high as 95.2%after the PBMNCs were cultured for ten days.After 24 h of co-cultured withγδTcells at the rario of 10:1 and 20:1,the proliferation of myeloma cells was significantly lower than the cells cultured alone(p<0.01).The flow cytometry results showed that the percentage of myeloma cells in G1-phase increased,and the percentage of cells in S-phase and G2-phase decreased significantly after co-cultivation withγδT cells.Co-cultivation withγδT cells could also induce more apoptotic cells in myeloma cells.These effects were dependent on the ratio ofγδT cells to myeloma cells.The expressions of cell cycle-related proteins Cyclin D1,CDK4 and CDK1 was significantly lower in myeloma cells of co-cultured groups than in control group.Detection of apoptosis-related proteins showed that the expressions of pro-apoptotic proteins Bax and cleaved caspase3 was significantly higher and that of anti-apoptotic protein Bcl-2 was significantly lower in myeloma cells of co-cultured group than in control group.Compared with control group,the expression of autophagy-related protein LC3B was significantly higher and autophagy degradation related protein P62 of was significantly lower in co-cultured groups. Western blotting of autophagy-related pathway proteins found that in myeloma cells of co-cultured group,the expression of PI3K,AKT,P-AKT and P-m TOR were significantly decreased.However,AMPK and Beclin-1 were significantly increased(p<0.05).The ELISA results showed that the concentration of IFN-γand TNF-αwas significantly higher in co-cultured groups than in control group(p<0.05).Conclusions:1.γδT cells can be expanded with Zol and IL-2 in vitro.2.γδT cells might block myeloma cells in G1 phase and inhibit the proliferation of myeloma cells.3.γδT cells might promote apoptosis of myeloma cells.4.γδT cells might increase the level of autophagy in myeloma cells by inhibiting the pathway of PI3K/AKT/m TOR and activating the pathway of AMPK/Beclin-1.5.The effect ofγδT cells promoting myeloma cells apoptosis and autophagy,and inhibiting myeloma cells proliferation may be associated with their release of IFN-γand TNF-α.