Anti-tumor Effects Of γδT Cells With Different Induction Systems And Different Growing Times On Lung Cancer A549 Cell Line

Objective To test the killing effect of human γδT cells prepared with different culture systems and different culture times on the lung cancer A549 cells line.Methods Isolated mononuclear cells(PBMC)from health adult peripheral blood.Activated the PBMC with zoledronic acid(5mmol/l),recombinant human interleukin-2(1000IU/ml),and CD3(50ng/ml)to expand human γδT cells in vitro.According to the different expansion systems of γδT cells,they were divided into group A(IL-2+zoledronic acid)and group B(IL-2+CD3+zoledronic acid).Grown for10,12,14 and 16 days,designed as A10,A12,A14 and A16;B10,B12,B14 and B16.The different ratios of effectors to targets(20:1 and 10:1),after 24 hours of co-incubation,(1)The killing of the prepared γδT cells to cancer cell line A549 was measured with MTT.(2)The cell markers(TCR-γδ,CD44,CD107 a and CD3)of the expanded γδT cells were analyzed with flow cytometry.Results(1)When the ratio of the T cells to the cancer cells(20:1),the killing rates ofγδT cells to A549 cell line in A10,A12,A14 and A16 are(67.62±2.32)%,(65.89±3.54)%,(75.88±2.43)% and(48.42±2.05)% respectively.In the group B,B10,B12,B14 and B16,the killing rates were(72.89±1.88)%,(75.84±1.02)%,(90.35±3.51)% and(56.33±0.62)% respectively.When the ratio of the T cells to the cancer cells(10:1),the killing rates of γδT cells to A549 cell line in A10,A12,A14 and A16 are(62.66±3.02)%,(57.68±3.29)%,(78.85±2.42)% and(46.72±0.27)%respectively.In the group B,B10,B12,B14 and B16,the killing rates were(72.19±1.70)%,(69.99±1.53),(89.78±2.68)% and(64.80±0.13)%.(1)Comparison between the different systems at the same culture time: The killing rate of group B in each culture period was significantly higher than that of group A(p<0.05).(2)Comparison between the same culture system and different expansion time,the killing activity of the 14 days prepared γδT cells in both systems,the activity was the strongest(p<0.05).When the expansion time was prolonged to 16 days,the anti-tumor of the activated immune cells in both groups decreased(p<0.05).Compared with other expansion times,its tumor-killing activity was the weakest in the 16 days-induction(p<0.05).The results showed that the γδT cells prepared by the two culture systems had the strongest tumor-killing activity 14 day-expansion.(2)The bio-markers of γδ T cells in group A and group B were analyzed by flow cytometry,and the same system was grown for different time: Group A:(1)A10,A12,A14 and A16 TCR-γδ surface markers were(91.50±3.07)%,respectively,(95.16±5.07)%,(96.14±5.07)% and(95.12±3.14)%,there is no significant differences between different time groups(p>0.05).(2)The expression of CD44 at different times are(99.07±0.25)%,(99.30±0.33)%,(99.16±0.26)% and(99.12±0.26)%,respectively,there was no significant difference on their expression(p>0.05).(3)The expression of CD107 a on the immune cells was(A10: 19.60±6.15)%,(A12: 3.62±0.47)%,(A14:1.54±0.03)%,and(A16: 3.75±0.38)% respectively,A10 had the highest expression rate(p<0.05).(4)In the group A,the expression of CD3 was(99.02±0.18)%,(98.97±0.34)%,(99.06±0.10)% and(99.26±0.26)% at different culture times,there was no significant differences(p>0.05).In the Group B:(1)The TCR-γδ bio-marker ofγδ T cells in B10,B12,B14 and B16 were(86.56±3.91)%,(94.07±3.67)%,(98.49±0.73)% and(97.77±1.99)% respectively.The expression rate of TCR-γδsurface marker in B16 cells was the highest(p<0.05).(2)The expression rate of CD44,in B10,B12,B14 and B16 was(98.82±0.26)%,(98.90±0.24)%,(98.94±0.34)% and(99.15±0.35)% respectively;CD107a expression rate was(72.62±3.97)%,(74.51±4.16)%,(76.16±6.39)% and(75.75±5.68)%;CD3 positive rate was(99.16±0.26)%,(99.07±0.14)%,(99.32±0.21)% and(98.59±0.61)% respectively.There was no significant difference between the time groups(p>0.05).At the same culture time,comparison between different culture systems:(1)The expression of TCR-γδ,CD44,and CD3 had no significant differences(p>0.05).(2)CD107a positive rate in the same growing time group,the B group was significantly higher than that in the A group(p<0.05).Conclusion(1)Within a period of the expansion,the prepared-γδT cells both group A(IL-2+zoledronic acid)and group B(IL-2+zoledronic acid+CD3)had strong killing effects on lung cancer A549 cells.However,if γδT cells were grown for too long,the killing activity of γδT cells could decrease.Experiments data confirmed that γδT cells cultured for 14 days had the strongest anti-tumor activity.The γδT cells induced and cultured in group B have stronger killing effect on lung cancer A549 cells than that in the group A.(2)Expansion time did not affect on the expression of TCR-γδ in the immune cells in the group,but its positive rate in the group B was the highest in 14 days.There were no significant differences between CD44 and CD3 in the preparation systems of A and B;However,CD107 a positive rate in the group A was much higher in the10 days expansion,.In the corresponding time of the two preparation systems,there was no significant difference in TCR-γδ,CD44 and CD3.But CD107 a positive cells in the group B were significantly higher than that of group A.