Killing Effect Of NKT Cells Obtained With Different Expansion Systems And Induction Times On A549 Cell Line

Objective: Using healthy human peripheral blood mononuclear cells,NKT cells were obtained with different expansion systems and culture times,to test their killing effects on cancer cell lines.Methods: Four samples of peripheral blood from healthy adults were collected under sterile conditions,and the mononuclear cells(PBMCs)were obtained by Ficoll density gradient centrifugation.The NKT cell culture systems were divided into group A(IL-2+IL-15)group and group B(IL-2 +IL-15+CD3 antibody).NKT cells were harvested after expansion in vitro to the 14,17,and 20 days,mixed with A549 cells at ratios of 20:1 and 10:1,then grown in 96-well plates.Designed as effector cell wells(NKT cells),target cell wells(A549 cells),and experimental group(NKT cells + A549 cells).Group A is marked as: NKT-A1,NKT-A2,A549,NKT-A1*,NKT-A2* groups and group B was designed as: NKT-B1,NKT-B2,A549 cells,NKT-B1*,NKT-B2* groups,each group had been repeated for 6 times and incubated for a total of 24 hours.Measure the absorbance(A)value at 490 nm of the cells in each well with an enzyme-linked immunoassay,recorded the data,and calculated the killing activity of each group of NKT cells with the formula.Cell cycle and surface markers of the prepared NKT cells analysed with flow cytometry.Then,SPSS 25.0 statistical software was used to analyze the data.Results:(1)Group A: After NKT cells grown in vitro for 14,17,20 days and lung cancer A549 cells were co-cultured for 24 hours,the killing activity with an effective target ratio of 20:1 was(78.930±4.194)%,(97.978±0.968),and(78.758±9.116)% respectively;the killing activity with an effective target ratio of 10:1 was(67.860±6.373)%,(87.678±5.836)% and(69.013±7.929)% respectively.The killing activities of group B with an effective-to-target ratio of 20:1 was(76.943±5.455)%,(93.965±1.693)% and(85.885±1.991)% respectively;the killing activities with an effective-to-target ratio of 10:1 was(71.751±3.836)%,(85.210±3.639)% and(74.135±6.591)% respectively.The NKT cells obtained by the two culture methods showed different degrees of killing effect on tumor cells,but there was no significant difference between the two culture systems of group A and group B(P>0.05),except that the effective target ratio of group B was at 10:1,there was no significant difference in killing activity between the 17 th day and the 20 th day of culture(P>0.05).The other experimental groups had significant differences between the two groups at different culture times(P<0.05).Among them,the NKT cell killing activity was the highest on the 17 th day of in vitro culture;in the case of two different effective target ratios,there was a significant difference in pairwise comparison between each experimental group(P<0.05).(2)Flow cytometry was employed to detect the cell cycle of the obtained NKT cells at different grown times,namely G0/G1,G1/M,and S phase.There was no significant difference between the two comparisons at 14,17,and 20 days(P>0.05).(3)NKT cells obtained from the two culture systems were tested for the expression of surface markers CD3+CD56+,CD3,and C56 at different culture times.There were significant differences on the 14,17,and 20 days compared with the 0 day(P< 0.05).The surface markers of NKT obtained at other different incubation times were similar(P>0.05).Conclusion:(1)The NKT cells obtained both in group A and group B could kill tumor cells.The killing activity was the highest on the 17 th day of culture in vitro.In addition,the killing activity was stronger when the ratio of effect to target at 20:1.(2)The NKT cells obtained in two expansion groups both could kill tumor cells,but the effect of CD3 antibody on enhancing the toxicity of NKT cells was not observed.(3)Peripheral blood-derived NKT cells,the expression of surface markers CD3+CD56+,CD3,and CD56 were all up-regulated with prolonging culture.(4)At different incubation times,the cell cycle of NKT cells expanded in group A and B had no significant difference.